Modified monoterpene indole alkaloid production in the yeast Saccharomyces cerevisiae

Abstract

Alkaloids are a large group of plant natural products that have important therapeutic value. Because of their chemical complexity, therapeutic alkaloids are often obtained via plant-extraction. The microbial synthesis of alkaloids would allow for their rapid and scalable production; however, often times microbial production of a modified natural product can be more beneficial than production of the natural product itself. Here, an analysis was completed of current alkaloid-based pharmaceuticals to determine the steps necessary to engineer microbes to produce beneficial modified alkaloids. The yeast Saccharomyces cerevisiae was engineered to produce the modified monoterpene indole alkaloid (MIA) hydroxystrictosidine, which could enable the accelerated semisynthesis of anti-cancer pharmaceuticals. The work presented in this thesis represents the first production of a modified MIA in S. cerevisiae. This achievement involved the use of a pterin-dependent mono-oxidation strategy for the microbial synthesis of the biogenic amine serotonin and the leveraging of serotonin to produce hydroxystrictosidine. Subsequently, a G-protein-coupled receptor-based sensor was developed to detect serotonin in the spent medium of a serotonin-producing microbe in a medium-throughput fashion in order to accelerate screening of improved serotonin producers, as an improved serotonin producer could then be converted into an improved modified MIA producer. Lastly, as compartmentalization of metabolic pathway enzymes can aid in improving production yields, the translocation efficiencies of multiple translocation tags were quantified to enable rational selection of translocation tags. Overall, this thesis presents the foundation for low-cost, renewable production of modified MIAs in the yeast S. cerevisiae.