IMPROVING ACCESS TO MALARIA TREATMENT AND PREVENTION MEASURES VIA BIOSENSOR-GUIDED EVOLUTION - PROJECT SUMMARY TERPENES, OTHERWISE KNOWN AS ISOPRENOIDS, ARE A LARGE AND DIVERSE CLASS OF NATURAL PRODUCTS THAT DISPLAY A VARIETY OF BIOLOGICAL ACTIVITIES. TWO SUCH MOLECULES, NOOTKATONE AND ARTEMISININ, PLAY CRITICAL ROLES IN BOTH THE PREVENTION AND TREATMENT OF ONE OF THE MOST DEVASTATING INFECTIOUS DISEASES: MALARIA. NOOTKATONE HAS BEEN PROVEN TO BE A POTENT INSECT REPELLENT, RIVALING THE PROPERTIES OF HARSH SYNTHETIC INSECTICIDES SUCH AS DEET. ARTEMISININ HAS BEEN USED FOR YEARS IN THE TREATMENT OF MALARIA AS A PART OF ARTEMISININ COMBINATION THERAPIES, SAVING BILLIONS OF LIVES. HOWEVER, DESPITE THE IMPORTANCE OF THESE MOLECULES, THE MAIN SOURCE OF PRODUCTION REMAINS PLANT EXTRACTIONS. THE NATURAL ABUNDANCE OF TERPENES IS EXTREMELY LOW AND OFTEN RESULTS IN POOR EXTRACTION EFFICIENCY. THIS, COUPLED WITH ANY VARIATIONS IN GROWTH CONDITIONS, CROP DISEASE, AND THE GEOPOLITICAL CLIMATE, CAN IMPACT THE AVAILABILITY OF THESE INVALUABLE PRODUCTS. TO CIRCUMVENT THESE ISSUES, THE USE OF MICROBES AS HIGH-YIELDING PRODUCTION FACILITIES SHOULD BE ESTABLISHED, THUS ENSURING THE ACCESSIBILITY OF BOTH NOOTKATONE AND ARTEMISININ. HOWEVER, VARIOUS BOTTLENECKS IN THESE MICROBIAL BIOSYNTHETIC PATHWAYS HAVE RESULTED IN LOW YIELDS OR HIGH PRODUCTION COSTS, PREVENTING THIS METHOD FROM BEING USED ON A LARGE SCALE. THE PROCESS OF DIRECTED EVOLUTION, WHERE RANDOM MUTATIONS ARE INTRODUCED TO THE RATE-LIMITING ENZYME IN THE BIOSYNTHETIC PATHWAY, CAN BE USED TO MIMIC HOW NATURE OPTIMIZES BIOSYNTHESIS. HOWEVER, THIS METHOD GENERATES THOUSANDS OF SAMPLES TO SCREEN. THE LACK OF ROBUST, HIGH-THROUGHPUT METHODS FOR SCREENING TERPENES HAS SEVERELY HINDERED THE ABILITY TO APPLY DIRECTED EVOLUTION TO ENHANCE THE MICROBIAL PRODUCTION OF TERPENES SUCH AS NOOTKATONE AND ARTEMISININ. TO ADDRESS THIS, THE OBJECTIVE OF THIS PROPOSAL IS TO DEVELOP GENETICALLY ENCODED BIOSENSORS THAT DETECT NOOTKATONE AND ARTEMISININ AND TO LEVERAGE THEM FOR HIGH- THROUGHPUT SCREENING OF ENZYME/PATHWAY LIBRARIES TO OVERCOME COMPLEX BOTTLENECKS AND IDENTIFY IMPROVED PRODUCTION STRAINS. IN THIS PROPOSAL, THE MACROLIDE-SENSING TRANSCRIPTION FACTOR MPHR WILL SERVE AS A TEMPLATE FOR THE DEVELOPMENT OF A TERPENE BIOSENSOR, BASED ON PRELIMINARY RESULTS INDICATING THAT MPHR VARIANTS CAN DETECT SEVERAL SESQUITERPENES. THE GOAL OF THE FIRST AIM IS TO ENHANCE THE IN VIVO PRODUCTION OF NOOTKATONE BY DIRECTED EVOLUTION OF CYTOCHROME P450 ENZYMES IN E. COLI, UTILIZING A GENETICALLY ENCODED BIOSENSOR TO REPORT THE P450- CATALYZED HYDROXYLATION OF VALENCENE. THE GOAL OF THE SECOND AIM IS TO TRANSFER THE E. COLI-BASED BIOSENSOR CAPABILITIES INTO YEAST AND DEVELOP A YEAST-BASED ARTEMISININ BIOSENSOR. THIS TOOL CAN THEN BE USED TO EVOLVE THE FIRST-EVER IN VIVO PRODUCTION OF ARTEMISININ. THE IMPACTS OF THE WORK DESCRIBED HERE NOT ONLY CONTRIBUTE SUBSTANTIALLY TOWARDS THE FIGHT AGAINST MALARIA BUT CAN ALSO IMPACT THE DEVELOPMENT OF OTHER PHARMACEUTICALLY RELEVANT TERPENES, SUCH AS THE ANTI-CANCER THERAPY TAXOL.