Not listed COLLABORATIVE RESEARCH: POISE UNDER PRESSURE: DEVELOPING STRAINS WITH MINIMAL GENOMES FOR INTEGRATED BIOPROCESSING -THE CURRENT CHALLENGES OF DEPENDENCE ON NON-RENEWABLE FOSSIL SOURCES TO GENERATE THE CHEMICALS AND MATERIALS THAT HUMANS DEPEND UPON, ALONG WITH THE ENERGY- AND CARBON-INTENSIVE PROCESSES THAT TRANSFORM THEM, MOTIVATES THE DEVELOPMENT OF NEW APPROACHES TO CONVERT RENEWABLE FEEDSTOCKS TO USEFUL PRODUCTS. ADVANCES IN BIOTECHNOLOGY HAVE RESULTED IN SUBSTANTIAL GROWTH WITHIN THE METHODOLOGIES AND COMMERCIAL ACTIVITIES THAT COMPRISE THE BIOECONOMY, INCLUDING THOSE WITH A FOCUS ON THE CONVERSION OF SUSTAINABLE FEEDSTOCKS. HOWEVER, IT REMAINS THE CASE THAT THE COSTS OF MANY BIO-BASED PRODUCTS ARE NOT SUFFICIENTLY COMPETITIVE TO DISPLACE FOSSIL-DERIVED COUNTERPARTS. IT IS ALSO TRUE THAT THE SCOPE OF PRODUCTS ACCESSIBLE THROUGH BIOLOGICAL PRODUCTION IS FREQUENTLY CONSTRAINED BY THE SENSITIVITY OF COMMONLY-USED MICROBES TO THE COMPOUNDS THAT ARE DESIRED TO BE PRODUCED. FINALLY, BECAUSE CONTAMINATION IS A SIGNIFICANT CONCERN, BIOPROCESSES ARE USUALLY OPERATED IN BATCH OR FED-BATCH MODE, WHILE CHEMICAL PROCESSES BENEFIT FROM THE PRODUCTIVITY ADVANTAGES INHERENT TO CONTINUOUS PRODUCTION. THIS PROJECT FOCUSES ON THE DEVELOPMENT OF A PARTICULAR ORGANISM THAT DISPLAYS AN UNUSUAL SET OF PHYSICAL CHARACTERISTICS TO HELP ADDRESSES THESE CHALLENGES. THE MICROBE IS CAPABLE OF GROWTH IN A TWO-PHASE SYSTEM THAT INCLUDES SUPERCRITICAL CO2 (SCCO2), WHICH IS A PREFERENTIAL SOLVENT FOR MANY TOXIC PRODUCTS AS WELL AS INHIBITS GROWTH OF MOST ORGANISMS. THIS SECONDARY PHASE SHOULD BOTH PROTECT THE MICROBE FROM HIGH CONCENTRATIONS OF THE PRODUCT THROUGH CONTINUOUS WITHDRAWAL, AND MINIMIZE THE RISKS OF CONTAMINATION. THIS RESEARCH PROJECT WILL RESULT IN GREATER UNDERSTANDING OF THE BEHAVIOR OF THIS ORGANISM, AN INCREASE IN GENETIC ENGINEERING TOOLS, AND A STREAMLINED STRAIN, ALL OF WHICH WILL LEAD TO NEW OPPORTUNITIES FOR INTEGRATED BIOPROCESS OPERATIONS, WHICH COUPLE PRODUCTION WITH EXTRACTION. THE WORK WILL ENABLE THE TRAINING OF GRADUATE STUDENTS AND POSTDOCTORAL RESEARCHERS AS WELL AS RESEARCH EXCHANGES BETWEEN THE COLLABORATING INSTITUTIONS. NEW CONTENT WILL ALSO BE INTRODUCED INTO AN EXPERIMENTAL LABORATORY CURRICULUM. THE GOAL OF THIS PROJECT IS TO ELUCIDATE THE GENOTYPE-PHENOTYPE RELATIONSHIP OF TOLERANCE TO SCCO2 OF THE CHOSEN MICROORGANISM USING A REDUCED GENOME APPROACH. THE OBJECTIVES INCLUDE THE DETERMINATION OF GENE ESSENTIALITY USING TRANSPOSON LIBRARIES, DEVELOPMENT OF ROBUST TOOLS FOR GENOME-SCALE ENGINEERING OF THE ORGANISM, AND CONSTRUCTION OF A MINIMAL GENOME STRAIN FOR USE IN BIOPRODUCTION. TRANSPOSON LIBRARIES WILL BE CONSTRUCTED FOR BOTH GENE KNOCKOUTS (I.E., COMPLETE ELIMINATION OF ASSOCIATED ENZYME ACTIVITIES) AND OVEREXPRESSION, AND THE RESULTING LIBRARIES WILL BE ASSESSED TO ESTABLISH ESSENTIALITY OF GENE SETS UNDER DIFFERENT ENVIRONMENTAL CONDITIONS. TRANSCRIPTOMIC (RNA SEQUENCING) AND TRANSLATOMICS (RIBOSOME PROFILING) STUDIES WILL BE PERFORMED ON LIBRARY MEMBERS TO UNDERSTAND SYSTEMATIC BIOLOGICAL RESPONSES TO CHANGES IN CULTURE CONDITIONS AT THE MOLECULAR LEVEL. THE ASSOCIATED DATA WILL ALSO BE USED TO MINE AND DESIGN NEW BIOPARTS TO EXPAND A TOOLBOX FOR GENETIC ENGINEERING. LASTLY, A REDUCED GENOME STRAIN DISPLAYING THE DESIRED TOLERANCE PHENOTYPE WILL BE CONSTRUCTED AND ENGINEERED TO PRODUCE TARGET COMPOUNDS, WITH THESE STRAINS EVALUATED RELATIVE TO THE WILD-TYPE PREDECESSOR. THIS PROJECT WILL PROVIDE DEEPER UNDERSTANDING OF TOLERANCE TO TOXIC SOLVENTS AND PROVIDE A WORKFLOW FOR ASSESSING THESE COMPLEX PHENOTYPES. IT IS THE NEXT STEP IN A LONG-TERM EFFORT TO INTRODUCE NEW, NON-MODEL ORGANISMS THAT ARE INHERENTLY ADVANTAGED FOR BIOPROCESSING, WITH THE ULTIMATE GOAL OF ADVANCING A ROBUST, SUSTAINABLE BIOECONOMY. 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. $0 7/23/26 Not listed COLLABORATIVE RESEARCH: POISE UNDER PRESSURE: DEVELOPING STRAINS WITH MINIMAL GENOMES FOR INTEGRATED BIOPROCESSING -THE CURRENT CHALLENGES OF DEPENDENCE ON NON-RENEWABLE FOSSIL SOURCES TO GENERATE THE CHEMICALS AND MATERIALS THAT HUMANS DEPEND UPON, ALONG WITH THE ENERGY- AND CARBON-INTENSIVE PROCESSES THAT TRANSFORM THEM, MOTIVATES THE DEVELOPMENT OF NEW APPROACHES TO CONVERT RENEWABLE FEEDSTOCKS TO USEFUL PRODUCTS. ADVANCES IN BIOTECHNOLOGY HAVE RESULTED IN SUBSTANTIAL GROWTH WITHIN THE METHODOLOGIES AND COMMERCIAL ACTIVITIES THAT COMPRISE THE BIOECONOMY, INCLUDING THOSE WITH A FOCUS ON THE CONVERSION OF SUSTAINABLE FEEDSTOCKS. HOWEVER, IT REMAINS THE CASE THAT THE COSTS OF MANY BIO-BASED PRODUCTS ARE NOT SUFFICIENTLY COMPETITIVE TO DISPLACE FOSSIL-DERIVED COUNTERPARTS. IT IS ALSO TRUE THAT THE SCOPE OF PRODUCTS ACCESSIBLE THROUGH BIOLOGICAL PRODUCTION IS FREQUENTLY CONSTRAINED BY THE SENSITIVITY OF COMMONLY-USED MICROBES TO THE COMPOUNDS THAT ARE DESIRED TO BE PRODUCED. FINALLY, BECAUSE CONTAMINATION IS A SIGNIFICANT CONCERN, BIOPROCESSES ARE USUALLY OPERATED IN BATCH OR FED-BATCH MODE, WHILE CHEMICAL PROCESSES BENEFIT FROM THE PRODUCTIVITY ADVANTAGES INHERENT TO CONTINUOUS PRODUCTION. THIS PROJECT FOCUSES ON THE DEVELOPMENT OF A PARTICULAR ORGANISM THAT DISPLAYS AN UNUSUAL SET OF PHYSICAL CHARACTERISTICS TO HELP ADDRESSES THESE CHALLENGES. THE MICROBE IS CAPABLE OF GROWTH IN A TWO-PHASE SYSTEM THAT INCLUDES SUPERCRITICAL CO2 (SCCO2), WHICH IS A PREFERENTIAL SOLVENT FOR MANY TOXIC PRODUCTS AS WELL AS INHIBITS GROWTH OF MOST ORGANISMS. THIS SECONDARY PHASE SHOULD BOTH PROTECT THE MICROBE FROM HIGH CONCENTRATIONS OF THE PRODUCT THROUGH CONTINUOUS WITHDRAWAL, AND MINIMIZE THE RISKS OF CONTAMINATION. THIS RESEARCH PROJECT WILL RESULT IN GREATER UNDERSTANDING OF THE BEHAVIOR OF THIS ORGANISM, AN INCREASE IN GENETIC ENGINEERING TOOLS, AND A STREAMLINED STRAIN, ALL OF WHICH WILL LEAD TO NEW OPPORTUNITIES FOR INTEGRATED BIOPROCESS OPERATIONS, WHICH COUPLE PRODUCTION WITH EXTRACTION. THE WORK WILL ENABLE THE TRAINING OF GRADUATE STUDENTS AND POSTDOCTORAL RESEARCHERS AS WELL AS RESEARCH EXCHANGES BETWEEN THE COLLABORATING INSTITUTIONS. NEW CONTENT WILL ALSO BE INTRODUCED INTO AN EXPERIMENTAL LABORATORY CURRICULUM. THE GOAL OF THIS PROJECT IS TO ELUCIDATE THE GENOTYPE-PHENOTYPE RELATIONSHIP OF TOLERANCE TO SCCO2 OF THE CHOSEN MICROORGANISM USING A REDUCED GENOME APPROACH. THE OBJECTIVES INCLUDE THE DETERMINATION OF GENE ESSENTIALITY USING TRANSPOSON LIBRARIES, DEVELOPMENT OF ROBUST TOOLS FOR GENOME-SCALE ENGINEERING OF THE ORGANISM, AND CONSTRUCTION OF A MINIMAL GENOME STRAIN FOR USE IN BIOPRODUCTION. TRANSPOSON LIBRARIES WILL BE CONSTRUCTED FOR BOTH GENE KNOCKOUTS (I.E., COMPLETE ELIMINATION OF ASSOCIATED ENZYME ACTIVITIES) AND OVEREXPRESSION, AND THE RESULTING LIBRARIES WILL BE ASSESSED TO ESTABLISH ESSENTIALITY OF GENE SETS UNDER DIFFERENT ENVIRONMENTAL CONDITIONS. TRANSCRIPTOMIC (RNA SEQUENCING) AND TRANSLATOMICS (RIBOSOME PROFILING) STUDIES WILL BE PERFORMED ON LIBRARY MEMBERS TO UNDERSTAND SYSTEMATIC BIOLOGICAL RESPONSES TO CHANGES IN CULTURE CONDITIONS AT THE MOLECULAR LEVEL. THE ASSOCIATED DATA WILL ALSO BE USED TO MINE AND DESIGN NEW BIOPARTS TO EXPAND A TOOLBOX FOR GENETIC ENGINEERING. LASTLY, A REDUCED GENOME STRAIN DISPLAYING THE DESIRED TOLERANCE PHENOTYPE WILL BE CONSTRUCTED AND ENGINEERED TO PRODUCE TARGET COMPOUNDS, WITH THESE STRAINS EVALUATED RELATIVE TO THE WILD-TYPE PREDECESSOR. THIS PROJECT WILL PROVIDE DEEPER UNDERSTANDING OF TOLERANCE TO TOXIC SOLVENTS AND PROVIDE A WORKFLOW FOR ASSESSING THESE COMPLEX PHENOTYPES. IT IS THE NEXT STEP IN A LONG-TERM EFFORT TO INTRODUCE NEW, NON-MODEL ORGANISMS THAT ARE INHERENTLY ADVANTAGED FOR BIOPROCESSING, WITH THE ULTIMATE GOAL OF ADVANCING A ROBUST, SUSTAINABLE BIOECONOMY. 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.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD. $153.5k 7/9/26