Project Grant 2222602

SBIR PHASE I: BOOSTING INDUSTRIAL BIO-FERMENTATION WITH MICROBIAL STEM CELLS -THE BROADER IMPACT OF THIS SMALL BUSINESS INNOVATION RESEARCH (SBIR) PHASE I PROJECT IS TO REIMAGINE BIO-MANUFACTURING WITH A NOVEL PLATFORM TECHNOLOGY THAT COULD BOOST THE YIELDS OF MANY PRODUCTS, INCLUDING FOOD ADDITIVES, BIOMATERIALS PRECURSORS, BIOFUELS, AND PHARMACEUTICALS. THE TECHNOLOGICAL ADVANCEMENT ADDRESSES A FUNDAMENTAL ISSUE THAT LIMITS CONVENTIONAL BIO-FERMENTATION, WHICH IS THAT PRODUCING CELLS SUFFER LIMITED HEALTH AND VIABILITY IN EXCHANGE FOR HIGHER YIELDS. IN THIS PROPOSAL, GENETIC TOOLS WILL BE USED TO DIVIDE THE LABOR OF CELL REPRODUCTION AND PRODUCT SYNTHESIS INTO TWO DIFFERENT CELL TYPES, CALLED STEM CELLS AND FACTORY CELLS. AS OLDER FACTORY CELLS BECOME EXHAUSTED, PRODUCTIVITY IS MAINTAINED BY NEW FACTORY CELLS, WHICH ARE BORN FROM THE STEM CELL POPULATION. THE APPROACH MAY BE PARTICULARLY WELL SUITED TO BIOFUELS AND OTHER MOLECULES THAT ARE DIFFICULT TO PRODUCE IN LARGE QUANTITIES BY CONVENTIONAL BIO-FERMENTATION BECAUSE THE PRODUCT IS TOXIC TO THE CELLS THAT MAKE IT. IT COULD BE APPLIED TOWARD INCREASING THE PROFITABILITY OF EXISTING BIO-PROCESSES AND ALSO FOR BRINGING NEW PRODUCTS TO MARKET, WHICH ARE CURRENTLY TOO DIFFICULT TO PRODUCE. IN THIS PROJECT, THE TEAM SEEKS TO DEMONSTRATE THE BENEFITS OF PRODUCING A FUEL (LIMONENE) AND A DAIRY ENZYME (CHYMOSIN), AS PROOF OF ITS APPLICATION IN BIOFUEL AND AGRICULTURAL SECTORS. BROAD INDUSTRIAL IMPLEMENTATION WILL ADVANCE BIO-MANUFACTURING TOWARD THE ?GREEN? REVOLUTION, CONTRIBUTING TO THE DEVELOPMENT OF CLEANER INDUSTRIES AND DECREASING US AND GLOBAL RELIANCE ON FOSSIL FUELS. THIS PROJECT AIMS TO SOLVE TWO MAJOR LIMITATIONS OF MICROBIAL FERMENTATION PROCESSES: METABOLIC EXHAUSTION AND GENETIC DRIFT. THESE ARE NEARLY UNIVERSAL PROBLEMS IN THE INDUSTRY. HIGHLY PRODUCING CELLS CAN BECOME INACTIVE DUE TO THE LACK OF METABOLIC RESOURCES, CYTOTOXIC EFFECTS OF PRODUCTS, AND MUTATIONS THAT BREAK THE BIOSYNTHETIC PATHWAY. IN THIS PROJECT, MICROBIAL STEM CELL TECHNOLOGY (MIST) UNCOUPLES GROWTH AND PRODUCTION BY ESTABLISHING A MULTICELLULAR SYSTEM. ONE CELL TYPE IS DEDICATED TO PRODUCT SYNTHESIS (FACTORY CELLS), WHILE ANOTHER (STEM CELLS) IS RESPONSIBLE FOR CELL DIVISION AND THE GENERATION OF NEW FACTORY CELLS. AS OLDER FACTORY CELLS LOSE PRODUCTIVITY, THE BIOREACTOR IS CONTINUOUSLY REPLENISHED WITH NEW FACTORY CELLS, DERIVED FROM THE STEM CELL POPULATION. BY MAINTAINING AN ACTIVE FACTORY CELL POPULATION, MIST-SUPPORTED CULTURES ARE EXPECTED TO EXHIBIT INCREASED PRODUCTION LONGEVITY AND HIGHER OVERALL YIELD THAN CONVENTIONAL BIO-FERMENTATIONS. THIS PROJECT AIMS TO VALIDATE THE TECHNOLOGY IN E. COLI ENGINEERED TO PRODUCE LIMONENE, A PRECURSOR FOR BIODIESEL AND OTHER USEFUL CHEMICALS. IN THE FACTORY CELLS, T7RNAP WILL DRIVE HIGH-LEVEL EXPRESSION OF A SUITE OF BIOSYNTHETIC ENZYMES. SINCE LIMONENE HAS A CYTOTOXIC EFFECT ON PRODUCING CELLS, MIST-SUPPORTED FACTORY CELL REPLENISHMENT IS EXPECTED TO INCREASE PRODUCTIVITY BY MORE THAN 2-FOLD COMPARED TO THE CONVENTIONAL LIMONENE-PRODUCING STRAINS. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.