Proposal Summary Kratom (Mitragyna speciosa) is a medicinal plant that has been used in Southeast Asia for hundreds of years to self-treat chronic-pain, opioid with-drawl, and depression. Due to these unique pharmacological effects, in the past decade Kratom has flourished in the U.S. with many users self-treating opioid use disorder and pain management. This medicinal herb has become a life-raft many use to pull themselves out of the pit of addiction, rather than continuously falling in the trap of relapsing. Despite the recent attention, this plant remains controversial in the eyes of the Food and Drug Administration and the Drug Enforcement Administration due to limited or preliminary clinical research. Although, several studies have been conducted on Kratom’s primary alkaloid mitragynine, characterizing it a partial antagonist of the μ-opioid receptor activated through the G-protein signaling cascade. Kratom users report pain relieving properties and a depletion in negative side effects such as constipation, addiction, and respiratory depression compared to morphine-based opioids. Furthermore, studies show that some mitragynine analogs such as mitragynine pseudoindoxyl and 7-hydroxymitragynine are even more potent agonists of the µ-opioid receptor, although a complete structure activity relationship of these alkaloids has not been established. Thus, unanswered questions remain regarding where and how to manipulate mitragynine to be the ultimate opioid substitution drug. Our goal is to fully elucidate the biosynthetic pathway of mitragynine to determine the biocatalytic transformations required to produce the mitragynine scaffold. Determining the biocatalytic pathway opens the door to manipulate critical catalysts that form mitragynine to produce analogs that may enhance the pharmacological properties already displayed from this unique alkaloid. Furthermore, we plan to derivatize mitragynine even further by characterizing and engineering flexible catalysts from other organisms to produce mitragynine analogs with alternative cyclization, stereochemistry or functional group patterns. Through this study we will generate a library of mitragynine analogs with the goal of identifying effective candidates to help treat opioid use disorder. The overreaching hypothesis is that through methods of protein engineering we can diversify the mitragynine scaffold beyond what is attainable through general synthetic approaches and prepare novel compounds with enhanced pharmacological properties concerning opioid response. Ultimately, mitragynine analogs have the potential to fast-track the development of new and safer treatments for opioid use disorders therefore permanently improving the lives of those affected.