The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to enhance the supply of complex pharmaceutical molecules from nature. Over 60% of current drugs are natural products, or are derived from natural products, and of these approximately half are from plants. The therapeutic activity of a given plant molecule is encoded in its chemical structure, which is biosynthesized by a specialized metabolic pathway. Despite the efficiency of these biosyntheses, plants accumulate relatively small amounts of the potent metabolites in specialized cells and tissue types, thereby restricting the availability of essential medicines from plants. The opioids exemplify this limitation of plant therapeutics; the structural complexity of the opioids precludes chemical synthesis at commercial scale, and in the absence of this synthetic source the only viable alternative is to extract natural opiates from opium poppy. This project proposes to make the biosynthesis of medicinal opioids possible in a microbial host. This synthetic biology approach will move opioid production into fermentation facilities and free up the 100,000 hectares of arable land used each year for poppy crops. The disruptive technology resulting from this research will for the first time provide for a local, scalable, secure supply of medicinal opioids. This SBIR Phase I project proposes to develop a microbial production system for medicinal opioids that will displace the existing supply from opium poppies. A complete opioid biosynthesis pathway was recently constructed in Baker's yeast, demonstrating that this technology holds enormous potential for supplying the $2B opioid active pharmaceutical ingredient (API) market. The key technical hurdle addressed in this SBIR project is to enhance the activity of rate-limiting enzymes that catalyze key steps in the construction of the five-ring opioid scaffold. The target class of plant enzymes is poorly expressed in heterologous hosts such as yeast and must be membrane localized. The proposed research takes three approaches to support these enzymes: 1) tuning expression to conserve the endomembrane environment and promote activity, 2) constructing N-terminal chimeric proteins with enhanced stability, and 3) identifying partner enzymes that support the catalytic function of these enzymes. The goal is to remove the bottleneck steps in existing production strains to allow for commercially-relevant titers of greater than 1 g/L. The outcome will be a new production system that offers active pharmaceutical ingredients at lower cost, with greater availability and variety of molecules, shorter lead times, and acute responsiveness to the medical demand for opioid therapeutics.
