THERE ARE ABUNDANT FOOD PROCESSING WASTES CONTAINING LARGE AMOUNTS OF CARBOHYDRATE AND OTHER ORGANICS. EXCEPT FOR LIMITED USE IN ANIMAL FEED, THESE FOOD PROCESSING WASTES OR BYPRODUCTS HAVE NOT BEEN USED EFFECTIVELY AND CAN CAUSE SERIOUS POLLUTION AND ECONOMIC BURDEN TO AGRICULTURAL AND FOOD INDUSTRIES. THIS PROJECT AIMS TO DEVELOP ENGINEERED STRAINS OF A YEAST-LIKE FUNGUS AUREOBASIDIUM PULLULANS FOR PMA AND MALIC ACID PRODUCTION FROM LOW-COST RENEWABLE SUBSTRATES SUCH AS SOY MOLASSES THAT ARE DIFFICULT TO USE IN CURRENT BIOREFINERIES. SOY MOLASSES IS A LOW-VALUE BYPRODUCT GENERATED IN MANUFACTURING SOY PROTEIN CONCENTRATE AND ISOLATE IN THE SOYBEAN PROCESSING INDUSTRY. SOY MOLASSES CONTAINS LARGE AMOUNTS OF RAFFINOSE-FAMILY OLIGOSACCHARIDES (RFO), WHICH ARE DIFFICULT TO DIGEST BY NON-RUMINANT ANIMALS AND THUS HAS LITTLE USE IN ANIMAL FEED. WITH THE ACCELERATING DEMAND FOR SOY PROTEIN CONCENTRATE AND ISOLATE AS AN ECONOMICAL AND HIGHLY NUTRITIOUS FOOD PRODUCT, THE GLOBAL SOY PROTEIN MARKET IS GROWING AT A 4.6% CAGR AND IS ANTICIPATED TO BE WORTH US $4 BILLION BY 2026. THERE IS AN URGENT NEED FOR THE SOYBEAN INDUSTRY TO FIND BETTER USE FOR THE LARGE SURPLUS AMOUNT OF SOY MOLASSES, WHICH OTHERWISE MUST BE TREATED BEFORE DISPOSAL DUE TO ITS HIGH ORGANIC (BOD) CONTENT.THIS PROJECT WILL DEVELOP A VERSATILE BUT LESS KNOWN/STUDIED BLACK YEAST A. PULLULANS FOR PMA, A NOVEL BIOPOLYMER, AND MALIC ACID (MA), A SPECIALTY AND PLATFORM CHEMICAL, PRODUCTION FROM AGRICULTURAL RESIDUES AND PROCESSING WASTES. A. PULLULANS HAS A LARGE GENOME WITH A LARGE NUMBER OF SECRETED PROTEINS, MANY OF WHICH ARE HYDROLASES CAPABLE OF DEGRADING PLANT MATERIALS. IT ALSO HAS THE ABILITY TO PRODUCE EXTRACELLULAR POLYMERS, INCLUDING PMA, WHICH HAS UNIQUE PROPERTIES DIFFERENT FROM THOSE OF MORE FAMILIAR AND WELL-STUDIED BIOPOLYMERS, INCLUDING POLY-LACTIC ACID (PLA), PHA, AND POLY-GLUTAMIC ACID (PGA). PMA HAS MANY POTENTIAL APPLICATIONS AS CARRIERS FOR DRUGS AND FLAVOR COMPOUNDS, AND ITS MONOMER (L-MALIC ACID) IS A POTENTIAL C4 PLATFORM CHEMICAL WITH A LARGE MARKET OF 200,000 TONS. HOWEVER, CURRENT PRODUCTION OF PMA AND MALIC ACID VIA CHEMICAL SYNTHESIS IS NOT SUSTAINABLE AND BIOLOGICAL PRODUCTION REQUIRES FURTHER STRAIN ENGINEERING TO IMPROVE FERMENTATION EFFICIENCY WITH ABUNDANT LOW-COST FEEDSTOCKS. THE PROPOSED RESEARCH WILL PROVIDE A BETTER UNDERSTANDING OF THIS FUNGUS IN ITS CARBON AND NITROGEN METABOLISM AND REGULATION ON CELL GROWTH AND PMA BIOSYNTHESIS, WHICH HAS NOT BEEN STUDIED BEFORE BUT IS ESSENTIAL TO BIOTECHNOLOGICAL APPLICATIONS OF THIS VERSATILE FUNGUS. IN ADDITION, THE NEW GENETIC AND METABOLIC ENGINEERING TOOLS AND STRAINS DEVELOPED IN THIS PROJECT WILL UNEARTH THE POTENTIAL OF THE METABOLICALLY VERSATILE A. PULLULANS AS A NOVEL HOST ORGANISM FOR VARIOUS BIOTECHNOLOGY APPLICATIONS, ESPECIALLY IN INDUSTRIAL PRODUCTION OF HIGH-VALUE NATURAL PRODUCTS SUCH AS PMA, PULLULAN, MELANIN, ANTIMICROBIAL COMPOUNDS, LIAMOCINS, AND VARIOUS ENZYMES.WHILE PMA CAN BE PRODUCED FROMLOW-COST SUBSTRATES, INCLUDING SUGARCANE JUICE, CANE MOLASSES, POTATO WASTE, SOY MOLASSES, JERUSALEM ARTICHOKE TUBER, CORN COB, CORN FIBER, WHEAT STRAW, AND SOYBEAN HULL, PRODUCT TITER, YIELD AND PRODUCTIVITY FROM LIGNOCELLULOSIC BIOMASS HYDROLYSATES ARE LOW AND MUST BE IMPROVED IN ORDER TO BE ECONOMICAL FOR INDUSTRIAL APPLICATION. THE POOR PERFORMANCE WITH THESE LOW-COST SUBSTRATES CAN BE ATTRIBUTED TO INHIBITION CAUSED BY SUGAR AND LIGNIN DEGRADATION PRODUCTS (E.G., 5-HYDROXYMETHYLFURFURAL, FURFURAL, ACETIC ACID, AND FORMIC ACID. ADAPTATION OF CELLS TO THE HYDROLYSATE COULD ALLEVIATE THE INHIBITION BY IMPROVING TOLERANCE TO TOXIC STRESS THROUGH, SUCH AS, THE ACTIVATED SULFUR ASSIMILATION PATHWAY. IN ADDITION, IMPURITIES PRESENT IN CRUDE SUBSTRATES, SUCH AS HEAVY METALS IN CANE MOLASSES, MIGHT ALSO INHIBIT CELL GROWTH AND PMA PRODUCTION, AND MUST BE REMOVED OR REDUCED TO A NON-TOXIC LEVEL. FURTHERMORE, PMA FERMENTATION WITH CRUDE SUBSTRATES CONTAINING MIXED SUGARS MIGHT BE LIMITED BYGLUCOSE-MEDIATED CRBON CATABOLITE REPRESSION. THESE ISSUES WILL HAVE TO BE EVALUATED AND RESOLVED ESPECIALLY WHEN LIGNOCELLULOSIC BIOMASS HYDROLYSATES AND CRUDE SUBSTRATES ARE USED FOR PMA FERMENTATION. ADAPTATION AND SCREENING FOR STRAINS FOR SPECIFIC SUBSTRATES THUS WILL BE CONDUCTED IN THIS PROJECT. RATIONAL METABOLIC ENGINEERING WILL ALSO BE CONDUCTED TO IMPROVE PMA YIELD (&GT;0.7 G/G SUGAR), TITER (&GT;50 G/L), AND REACTOR PRODUCTIVITY (&GT;1.0 G/L/H) FROM THESE SUBSTRATES, WHICH WILL GREATLY REDUCE THE PRODUCT COSTS TO AN ECONOMICAL LEVEL TO COMPETE EFFECTIVELY WITH CHEMICALLY SYNTHESIZED PRODUCTS.THE BIOPROCESS TO BE DEVELOPED FOR PMA AND MA PRODUCTION WILL HAVE ENERGY AND COST ADVANTAGES OVER CONVENTIONAL CHEMICAL SYNTHESIS OF MALIC ACID. THE PROPOSED PROCESS ALSO HAS AN EDGE OVER OTHER BIOPROCESSES SUCH AS THOSE BEING RESEARCHED/DEVELOPED FOR MALIC, SUCCINIC, AND FUMARIC ACIDS USING OTHER FUNGI AND BACTERIA (MAINLY E. COLI). IN GENERAL, ORGANIC ACIDS PRODUCED IN CONVENTIONAL FERMENTATION PROCESSES ARE DIFFICULT AND EXPENSIVE TO SEPARATE AND PURIFY FOR USE IN THE CHEMICAL MARKET DUE TO THE CO-PRODUCTION OF OTHER ORGANIC ACIDS. MALIC ACID PRODUCTION VIA PMA FERMENTATION USING A. PULLULANS CAN OVERCOME THIS SEPARATION ISSUE.AN EFFICIENT PMA AND MALIC ACID PRODUCTION PROCESS WILL BENEFIT THE ECONOMIC DEVELOPMENT AS WELL AS THE ENVIRONMENT BY PROVIDING BIOBASED HIGH-VALUE FUNCTIONAL BIOPOLYMER AND SPECIALTY AND PLATFORM CHEMICALS. WITH GREATLY REDUCED FEEDSTOCK COSTS, VIA THE USE OF LOW-COST FOOD PROCESSING WASTES SUCH AS SOY MOLASSES, AND SIMPLIFIED DOWNSTREAM PROCESSING, MALIC ACID CAN BE PRODUCED VIA PMA FERMENTATION AT LESS THAN $1.10/KG, WHICH IS SIGNIFICANTLY LOWER THAN THE CURRENT MARKET PRICE OF $1.75/KG. MALIC ACID IS THE SECOND MOST WIDELY USED ACIDULANT IN THE FOOD INDUSTRY. IN THE FOOD AND CONSUMER MARKETS, THE BIO-BASED MALIC ACID WILL COMPETE WITH MALIC ACID PRODUCED FROM FUMARIC ACID AND MALEIC ANHYDRIDE, WHICH ARE DERIVED FROM PETROLEUM-BASED FEEDSTOCK. AS A C4 PLATFORM CHEMICAL, THE BIO-BASED MALIC ACID WILL COMPETE IN THE CHEMICAL MARKET MAINLY WITH PETROLEUM-BASED MALEIC ANHYDRIDE AND FUMARIC ACID AND ALSO WITH OTHER C4 CHEMICALS SUCH AS SUCCINIC ACID. MALIC ACID ALSO CAN BE CHEMICALLY CONVERTED TO FUMARIC ACID AND MALEIC ANHYDRIDE, WHICH HAS A LARGE MARKET OF OVER 2 MILLION MT. THE BIO-BASED MALIC ACID AT A COMPETITIVE COST HAS THE POTENTIAL TO REPLACE PETROLEUM-DERIVED FUMARIC ACID AND MALEIC ANHYDRIDE. THE PROPOSED PROCESS NOT ONLY WILL BENEFIT THE SOYBEAN REFINERY INDUSTRY, BUT ALSO WILL HAVE LARGE IMPACTS ON ECONOMICS DEVELOPMENT IN THE RURAL (AGRICULTURAL) AREAS AS WELL AS THE ENVIRONMENT BY PROVIDING ENVIRONMENTALLY FRIENDLY AND SUSTAINABLE MALIC ACID PRODUCTION FROM LOW-COST AGRICULTURAL AND FOOD PROCESSING WASTES.