Tools and platforms to advance the engineering of yeast for plant natural product biosynthesis
Keywords: Chemical Engineering.

Plant natural products play a critical role in our healthcare systems. It is estimated that up to 25% of modern drugs are derived from plant natural products. However, the cultivation of plants to produce, harvest, and extract plant natural product drugs requires a significant investment of land, water, and energy. In addition, the production supply chain, which includes a lengthy plant growth step, can result in frequent supply shortages. Increasingly, researchers are looking to microbial hosts, such as yeast, as alternative heterologous production hosts for plant natural products. Yeast have short doubling times to generate biomass quickly, can be readily engineered using a variety of genetic manipulation tools, and are able to functionally express a diversity of complex proteins and enzymes that play a role in plant secondary metabolism. As a result, several biosynthetic pathways of clinical importance have been reconstructed in yeast over the past decade, with a number now scaling to commercial production. Examples of plant-derived medicines that have been produced in yeast include analgesics like thebaine and hydrocodone, antitussives like noscapine, and neuromuscular agents like hyoscyamine and scopolamine. While significant progress in engineering yeast to produce complex plant natural products has been made, several challenges remain. One key challenge is in the elucidation of the biosynthetic routes evolved in plants to produce these secondary metabolites. Pathway discovery workflows incorporating genome mining and RNA co-expression have made significant advances in elucidating biosynthetic pathways, but for many pathways, there are enzymes responsible for key conversion steps that remain unknown. Additionally, functional expression of the enzyme or protein in a microbial host can present further challenges. Controlling flux through long, multi-step heterologous pathways often presents another challenge to efficient yeast biosynthesis of plant natural products. Pathway intermediates can be diverted through native host metabolism or exported out of the host before being converted by the next enzyme in the pathway of interest. My thesis work focuses on the production of tetrahydropapaverine (THP) and papaverine. To-date the biosynthesis of THP and papaverine in a heterologous host not been achieved, in part because the full plant biosynthetic pathway has not been elucidated. THP and papaverine are BIAs with established clinical significance that are extracted from the opium poppy. THP is a precursor in the production of the neuromuscular blocking agents atracurium and cisatracurium. These drugs, often administered during anesthesia to facilitate intubation, have experienced recent global supply shortages. Papaverine is used directly in the clinic as a vasodilator and antispasmodic and similarly experienced supply shortages over the past decade. To reconstruct the THP biosynthetic pathway in yeast, we identified enzymes with similar activities to the unidentified enzymes in the native plant pathway and improved their activity on pathway intermediates using protein engineering strategies. We used a combination of random and semi-rational mutagenesis techniques to identify enzyme variants with significantly increased activity on the non-native substrates. We also increased the flux through the pathway by knocking out two native yeast transporters that affect the export of pathway intermediates. We then accomplished the semi-synthesis of papaverine by combining the THP biosynthesis route with a one-step, aqueous chemical oxidation reaction. This work describes the first de novo biosynthesis of THP and semi-synthesis of papaverine. The strategies we used to synthesize these products, despite multiple missing steps in the pathway, can be broadly implemented in plant natural product biosynthesis and semi-synthesis