FATTY ACID ESTERS, MANY OF WHICH HAVE FRUITY FLAVORS AND FRAGRANCES, ARE VALUABLE CHEMICALS WITH EXTENSIVE APPLICATIONS IN FOOD, BEVERAGE, COSMETICS, AND PHARMACEUTICAL INDUSTRIES. SHORT-CHAIN ESTERS CAN ALSO BE USED FOR SOLVENTS, COATINGS, PAINTS, AND FUEL COMPONENTS. IT IS PROJECTED THAT THE US MARKET DEMAND FOR FATTY ACID ESTERS WILL REACH $4.34 BILLION BY 2022. WHILE THE TRADITIONAL APPROACHES FOR ESTER PRODUCTION THROUGH PETROLEUM CHEMICAL ROUTES ARE HIGHLY ENERGY-CONSUMING AND GENERATE A LARGE AMOUNT OF ENVIRONMENTAL POLLUTANTS, MORE AND MORE INTERESTS HAVE BEEN EVOKED RECENTLY FOR ESTER PRODUCTION THROUGH BIOLOGICAL ROUTES. THERE ARE GENERALLY TWO MICROBIAL PATHWAYS FOR ESTER PRODUCTION, EITHER BY ALCOHOL ACYL TRANSFERASES (AATS) WITH ACYL-COA AND ALCOHOLS AS FEEDING COMPOUNDS, OR BY LIPASE WITH FATTY ACID AND ALCOHOL AS REACTING MATERIALS. NON-PATHOGENIC CLOSTRIDIUM HAS TREMENDOUS SIGNIFICANCE FOR INDUSTRIAL BIOCHEMICAL PRODUCTION. WITH THE ANAEROBIC FERMENTATION PATHWAY, IT CAN GENERATE ACYL-COAS (ACETYL-COA AND BUTYL-COA), ACIDS (ACETIC ACID AND BUTYRIC ACID), AND ALCOHOLS (ETHANOL AND BUTANOL). THEREFORE, IN THIS PROJECT, WE PROPOSE TO USE CLOSTRIDIUM AS A PLATFORM TO BE METABOLICALLY ENGINEERED FOR ESTER PRODUCTION, PARTICULARLY FOR BUTYL BUTYRATE (BB) PRODUCTION. FIRST, BASED ON THE CRISPR-CAS SYSTEM THAT WE RECENTLY DEVELOPED FOR GENOME ENGINEERING, WE WILL ENGINEER THE CLOSTRIDIUM STRAIN FOR ENHANCED BUTYRYL-COA/BUTYRATE AND BUTANOL CO-PRODUCTION, PROVIDING FEEDING COMPOUNDS FOR BB PRODUCTION. FURTHER, WE WILL ENGINEER THE MODIFIED STRAIN FOR BB PRODUCTION, BY INTRODUCING LIPASE GENES (PRODUCING BB FROM BUTYRIC ACID AND BUTANOL) AND AAT GENES (PRODUCING BB FROM BUTYRYL-COA AND BUTANOL). MEANWHILE, WE WILL DEVELOP A GENOME-SCALE METABOLIC MODEL, TO GUIDE OUR FURTHER METABOLIC OPTIMIZATION FOR ENHANCED BB PRODUCTION. ULTIMATELY, WE WILL ESTABLISH A BIOPROCESS BASED ON THE METABOLICALLY STABLE CLOSTRIDIUM STRAINS FOR RENEWABLE ESTER PRODUCTION FROM LOW-VALUE CARBON SOURCES.THIS PROJECT AIMS TO TACKLE A KEY ISSUE RELATED TO BIOFUEL/BIOCHEMICAL PRODUCTION BY PRODUCING A HIGH VALUE BIOPRODUCT THAT IS EASILY RECOVERABLE, THUS DECREASING ENDPRODUCTS TOXICITY AND IMPROVING FEEDSTOCK CONVERSION EFFICIENCY AND PRODUCT PRODUCTION RATE. THIS RESEARCH ALIGNS WELL WITH THE PRIORITY AREA OF BIOPROCESSING AND BIOENGINEERING. VALUE-ADDED BB WILL BE PRODUCED FROM LOW-VALUE MATERIALS THROUGH OPTIMIZED BIOPROCESS USING ENGINEERED MICROORGANISM. THIS REPRESENTS A POTENTIALLY TRANSFORMATIVE RESEARCH WHICH CAN LEAD TO AN ENABLING BIOPROCESS IN SUPPORT OF THE US BIOECONOMY. IT ALIGNS WELL WITH THE US ENDEAVORS TO FOSTER THE PRODUCTION CAPACITY OF BIOFUELS, BIOENERGY, AND BIOPRODUCTS FROM LOW-VALUE MATERIALS.