Chapter Iv: Production of (r, S)-reticuline and Downstream Benzylisoquinoline Alkaloids in Saccharomyces Cerevisiae

The benzylisoquinoline alkaloids (BIAs) are a diverse class of plant secondary metabolites that exhibit a broad range of pharmacological activities and are synthesized through biosynthetic pathways that exhibit complex enzyme activities and regulatory strategies. We have engineered yeast to produce the major branch point intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches. 75 4.