Synthetic biology to discover kratom natural product biosynthetic pathways for biased analgesic development

Project Summary Opioid alternative analgesics that would kill pain potently without the respiratory side effect will be of specific importance in addressing the opioid crisis. The medicinal herb kratom (Mitragyna speciosa) produces natural products (mitragynine and 7-hydroxymitragynine) that selectively activate analgesia only, which are promising “biased” opioid alternatives. Elucidating the kratom biosynthetic pathway composed of enzymes catalyzing sequential reactions toward the syntheses of mitragynine, 7-hydroxymitragynine, or other kratom products is important to the development of “biased” non-opioid analgesics. We propose to identify and reconstruct both the kratom biosynthetic pathways and the plant enzyme complexes organizing plant biosynthesis in yeast by integrating plant functional genomics analysis and synthetic biology. The research will provide new chemical and plant biology knowledge to a broader community, develop a post-translational level understanding of plant pathway regulation in yeast, and highlight the possibility of using synthetic biology to understand how nature achieves complicated natural product biosynthesis. This proposal will develop the conceptual framework to understand how nature organizes the specialized metabolism in plant toward high efficiency and complexity. The development of high-throughput protein-protein interaction method in yeast for plant complex identification will provide new methods and knowledge for plant pathway discovery and reconstruction. The methods and knowledge we develop toward these goals will also enable the structural elucidation, biological activity evaluation, and microbial production of biased opioid alternatives, leading to the development of next-generation painkillers to address the opioid crisis.