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Development of Genetically Engineered Stress Tolerant Ethanologenic Yeasts Using Integrated Functional Genomics for Effe

Development of Genetically Engineered Stress Tolerant Ethanologenic Yeasts Using Integrated Functional Genomics for Effective Biomass Conversion to Ethanol Z. Lewis Liu National Center for Agricultural Utilization Research, USDA-ARS. Z. Lewis Liu. Outlines. INTRODUCTION

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Development of Genetically Engineered Stress Tolerant Ethanologenic Yeasts Using Integrated Functional Genomics for Effe

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  1. Development of Genetically Engineered Stress Tolerant Ethanologenic Yeasts Using Integrated Functional Genomics for Effective Biomass Conversion to Ethanol Z. Lewis Liu National Center for Agricultural Utilization Research, USDA-ARS

  2. Z. Lewis Liu Outlines • INTRODUCTION • - Biomass Pretreatment Generates Fermentation Inhibitors • - Microbial Performance is the Key for Improvement • - Why Integrated Functional Genomics? • METABOLIC CONVERSION PATHWAYS • - Furfural • - HMF (5-Hydroxymethylfurfural) • > Identification of 2,5-bis-hydroxymethylfuran • > Biotransformation of HMF • ADAPTIVE RESPONSE • POTENTIAL FOR YEAST TOLERANCE IMPROVEMENT • - Enhanced Biotransformation • - Tolerance Improvement Potential • BYPRODUCT UTILIZATION ISSUES • UNDERSTANDING TOLERANCE MECHANISMS USING FUNCTIONAL GENOMICS • - Quality Control Development for Microarray Studies • - Genomic Expression Response • NOVEL STRAIN DESIGN AND GENETIC ENGINEERING

  3. Z. Lewis Liu Biomass Pretreatment Generates Fermentation Inhibitors 100+ potential inhibitory compounds Representative inhibitors Furfural HMF (5-Hydroxymethylfurfural) Glucose Cellulose HMF Biomass Dehydration Hemicellulose Furfural Xylose Arabinose

  4. Z. Lewis Liu Microbial Performance is the Key for Improvement • Fast Life Cycle • Genetic Diversity • Biocatalyst • Bioengine • Economic Cost

  5. Z. Lewis Liu Ethanol Fermentation Process Ethanol and Byproducts The Black Box? Input Materials Anaerobic Microbial Fermentation

  6. Ethanol Fermentation Process (continued) glucose 2 NAD+ 2 ADP glycolysis 2 ATP NAD+ 2 + 2H+ 2 NADH 2x pyruvate H+ NAD+ regeneration 2x acetaldehyde 2x CO2 2x ethanol Z. Lewis Liu

  7. Why Integrated Functional Genomics? • Life is under integrated control program • Single gene function not enough • Interactions and Intermediates • Key genes • Regulatory elements and dynamic controls Z. Lewis Liu

  8. Metabolic Conversion Pathways Furfural Furfural Furfuryl Alcohol HMF (5-Hydroxymethylfurfural) HMF HMF Alcohol? HMF other than HMF Alcohol Z. Lewis Liu

  9. Metabolic Conversion Pathways (cont.) Identification of 2,5-bis-hydroxymethylfuran HMF2,5-bis-hydroxymethylfuran Z. Lewis Liu

  10. Metabolic Conversion Pathways (cont.) Biotransformation of HMF Z. Lewis Liu

  11. Adaptive Response Z. Lewis Liu

  12. Potential for Tolerance Improvement Enhanced Tolerance Against HMF Enhanced Detoxification of HMF Z. Lewis Liu

  13. Enhanced Tolerance and Detoxification of Furfural Z. Lewis Liu Potential for Tolerance Improvement (cont.)

  14. Z. Lewis Liu Enhanced Biotransformation Enhanced Tolerance Enhanced Detoxification IMPROVEMENT POTENTIAL

  15. Byproduct Utilization Biomass Hydrolysates Bring New Ingredients into Fermentation Z. Lewis Liu

  16. Tolerance Mechanisms by Functional Genomics Quality Control Development for Microarray Studies Exogenous Nucleic Acid Controls Cab(photosystem I chlorophyll ab binding protein) MSG(major latex protein) RBS1(ribulose bisphosphate carboxylase small chain 1 precursor) Cy3Cy5 Z. Lewis Liu

  17. Z. Lewis Liu Tolerance Mechanisms by Functional Genomics Genomic Expression

  18. Z. Lewis Liu Genomic Expression (cont.)

  19. Genomic Expression (cont.) Repressed-Repressed Enhanced-Enhanced Enhanced-Repressed Z. Lewis Liu

  20. Genomic Expression (cont.) 1 2 3 4 5 6 7 8 9 Z. Lewis Liu

  21. Novel Strain Design and Genetic Engineering Functional Genomics Pathway Analysis Tolerant Strain Resources Stress Tolerance Mechanisms Biological Experimental Confirmation Gene Cloning In vitro Expression Fermentation Tests Biochemical Pathway Analysis Integrated Programming Novel Strain Design Hypothesis Development In situ Detoxification Recombinant DNA High Yield / Low Cost Robust strain Improvement Z. Lewis Liu

  22. Mission Objectives More Efficient Biomass Conversion to Ethanol Z. Lewis Liu

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