CRITICAL KNOWLEDGE GAPS EXIST REGARDING HOW LIPID SUBSTRATE METABOLISM OF THE INDUSTRIALLY IMPORTANT YEAST YARROWIA LIPOLYTICA CAN BE CHARACTERIZED AND TRANSFORMED INTO EFFECTIVE METABOLIC AND PROCESS ENGINEERING TECHNOLOGIES TO VALORIZE THE ESTIMATED 30 BILLION POUNDS OF WASTE LIPIDS PRODUCED BY THE US ANNUALLY. TO FILL THESE CRITICAL KNOWLEDGE GAPS TO ESTABLISH NEXT-GENERATION MICROBIAL BIOREFINERIES FOR VALORIZING WASTE LIPIDS, OUR OVERALL PROJECT OBJECTIVE IS TO DEVELOP NOVEL EXPERIMENTAL AND MODELING TOOLS TO GUIDE ENGINEERING OF Y. LIPOLYTICA FOR OVERPRODUCTION OF A MODEL HIGH-VALUE TERPENOID, ASTAXANTHIN, FROM LIPID SUBSTRATES, IN AN EXTRACTIVE FERMENTATION PROCESS. THE CENTRAL HYPOTHESIS IS THAT YARROWIA'S ENORMOUS LIPID CATABOLIC CAPACITY CAN BE DECODED AND REWIRED, GUIDED BY METABOLIC ANALYSIS AND MODELING, FOR ASTAXANTHIN OVER-PRODUCTION, WHILE THE SYNERGISTIC USE OF LIPID AS BOTH SUBSTRATE AND PRODUCT EXTRACTANT FURTHER ENHANCES FERMENTATION RATE AND TITER. BY FILLINGTHE CRITICAL KNOWLEDGE GAPS, IT PROVIDES THE FOUNDATION NECESSARY FOR ELUCIDATING YARROWIA LIPID-SUBSTRATE METABOLISM AND FOR DRIVING MODEL-ASSISTED STRAIN ENGINEERING AND BIOPROCESS DEVELOPMENT. THE FOLLOWING THREE SPECIFIC AIMS ARE PROPOSED: 1) DECIPHER LIPID ASSIMILATION AND FLUX NETWORK FOR ASTAXANTHIN BIOSYNTHESIS IN YARROWIA UTILIZING FATTY CARBON SOURCES; 2) ENGINEER LIPID-FED YARROWIA FOR ASTAXANTHIN OVERPRODUCTION; AND 3) DEVELOP LIPID-SUBSTRATE EXTRACTIVE FERMENTATION WITH ASTAXANTHIN-OVERPRODUCING YARROWIA STRAINS. THE PROPOSED PROJECT IS SIGNIFICANT AND ALIGNS WITH NIFA AFRI PRIORITIES BECAUSE IT IS EXPECTED TO ADVANCE BIOPROCESSING AND BIOENGINEERING TECHNOLOGIES THAT ENABLE EFFICIENT UTILIZATION OF AN ALTERNATIVE RENEWABLE FEEDSTOCK, I.E., WASTE LIPIDS DERIVED FROM FOOD AND AGRICULTURAL SYSTEMS, TO VALUE-ADDED BIO-BASED PRODUCTS LIKE HIGH-VALUE OLEOCHEMICALS.