High-throughput technologies for natural products biosynthesis in yeast cell factories

Synthetic biology tools have come a long way since the first endeavors at genetic engineering in the 1970s, and the model organism Saccharomyces cerevisiae, commonly known as baker’s yeast, has been the center stage for many of these tools to develop. This work leverages the ease of genetic engineering in yeast, and the similarity between the yeast pheromone sensingpathway and that of human G protein-coupled receptor (GPCR) signaling, to develop a pipeline through the early steps in drug discovery contained entirely in yeast and guided by virtual screening. De-novo production of the monoterpene indole alkaloids alstonine, serpentine, intermediates, and several yohimbanes is demonstrated in yeast. Lead discovery is then finalized through an assay for physiological relevance exemplified using a yeast biosensor for human GPCR interaction, showing that alstonine is an antagonist of a human adrenergic receptor. Chemical diversification, akin to common efforts in lead optimization, was achieved with halogen derivatization in yeast, and by combining iterative HPLC-fractionation and yeast-based GPCR screening, continued physiological relevance was determined for halogen
derivatives of yohimbanes and putative 6F-tetrahydroharman. Finally, a virtual screening method to aid GPCR target identification in future efforts is developed.