Abstract

Benzylisoquinoline alkaloids (BIAs) are a class of plant secondary metabolites with interesting pharmaceutical properties, such as analgesic, anti-cancer, anti-spasmodic, anti-microbial, anti-tussive, vasodilation, and muscle relaxant. The BIA family includes the opiates morphine and codeine. Several other BIAs are also commercially available; relevant to this thesis is sanguinarine, which is marketed as a growth enhancer for animal feed by the company Phytobiotics. To date, commercial production of almost all BIAs relies on their extraction from plants. However, the rarity of most BIAs in nature and the cost and difficulty of their chemical synthesis limits research and potential valorization of most of these compounds. An alternative route to BIA synthesis could enable sustainable production of BIAs currently extracted from plants while also expanding access to rarer alkaloids. Synthesis in a microbial host like baker’s yeast is an attractive option due to cheap feedstock, genetic tractability, and ease of scaleup. The highest BIA titer produced in a microbial host is currently 4.6 g/L of (S)-reticuline, the last common pathway intermediate to several commercially relevant alkaloids, including sanguinarine. This success has not yet been carried forward to downstream targets, whose reported titers remain at the microgram to low milligram scale.

This thesis is focused on the development of a microbial platform for the synthesis of the BIA dihydrosanguinarine, the direct precursor of sanguinarine. In 2014 our group demonstrated the reconstruction of a partial synthesis pathway in yeast, resulting in 1.5% conversion of the supplemented BIA norlaudanosoline to dihydrosanguinarine. Here, we improve conversion of norlaudanosoline to dihydrosanguinarine to 10% by eliminating a pathway bottleneck. Next, we introduce de novo norlaudanosoline synthesis to yeast, which improves yields and reduces side products compared to the route previously used for BIA synthesis. Finally, we extend reticuline synthesis to dihydrosanguinarine, demonstrating its first de novo production in a microbial host and culminating in 630 mg/L of dihydrosanguinarine and sanguinarine in fed-batch fermentation. This represents a >300-fold increase over the previous highest reported titer of a commercial BIA target – a major milestone in the quest to make the entire BIA family commercially accessible.