** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THE LEAVES OF MITRAGYNA SPECIOSA (KRATOM), PRODUCE MORE THAN 50 MONOTERPENE INDOLE ALKALOIDS (MIAS) AND SPIROOXINDOLE ALKALOIDS ASSOCIATED WITH VARIED PHARMACEUTICAL USES. FOR EXAMPLE, THE MIA MITRAGYNINE HAS SHOWN PROMISE AS A POTENTIAL TREATMENT FOR PAIN, OPIOID USE DISORDER, AND OPIOID WITHDRAWAL WITHOUT ANY DEMONSTRATED ADDICTION POTENTIAL; AND THE SPIROOXINDOLE MITRAPHYLLINE SHOWS ANTI-TUMOR ACTIVITY. THE EXISTING KNOWLEDGE GAP IN THE BIOSYNTHESIS OF BIOACTIVE M. SPECIOSA ALKALOIDS PREVENTS SYSTEMATIC ADVANCES IN ENGINEERED PRODUCTION OF THESE HIGH VALUE COMPOUNDS, CREATING A BOTTLENECK FOR PHARMACEUTICAL RESEARCH AND PRODUCTION. THIS PROPOSAL COMBINES EXPERTISE OF AN INTERNATIONAL INTERDISCIPLINARY TEAM TO ELUCIDATE THE BIOSYNTHESIS OF MULTIPLE BIOACTIVE ALKALOIDS IN MITRAGYNA SPECIES.A MULTI-FACETED APPROACH INCORPORATING GENOMICS, TRANSCRIPTOMICS, AND METABOLOMICS WILL BE JOINTLY APPLIED TO GENERATE GENOMIC RESOURCES FOR MITRAGYNA SPECIES, INCLUDING GENOME AND TRANSCRIPTOME ASSEMBLIES AND ANNOTATION TO ENABLE PATHWAY DISCOVERY OF DIVERSE BIOACTIVE ALKALOIDS; AND ELUCIDATE THE BIOSYNTHESIS OF SELECTED PHARMACEUTICAL MIAS AND SPIROOXINDOLE ALKALOIDS. THE METABOLIC ENGINEERING AND FURTHER BIOSYNTHETIC UNDERSTANDING OF MITRAGYNA ALKALOIDS ENABLED BY THIS PROJECT ARE EXPECTED TO HAVE A SIGNIFICANT POSITIVE IMPACT TOWARDS THE MITIGATION OF THE OPIOID CRISIS IN THE U.S AND AROUND THE WORLD. IN ADDITION, GENOMIC RESOURCES GENERATED IN THIS PROJECT WILL BE AN INVALUABLE RESOURCE FOR THE SCIENTIFIC COMMUNITY IN MIA AND SPIROOXINDOLE ALKALOIDS RESEARCH.