National Center for Agricultural Utilization Research, USDA-ARS 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
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 Z. Lewis Liu
Biomass Pretreatment Generates Fermentation Inhibitors 100+ potential inhibitory compounds Representative inhibitors Furfural HMF (5-Hydroxymethylfurfural) Cellulose Glucose HMF Biomass Dehydration Hemicellulose Furfural Xylose Arabinose Z. Lewis Liu
Microbial Performance is the Key for Improvement Fast Life Cycle Genetic Diversity Biocatalyst Bioengine Economic Cost Z. Lewis Liu
Ethanol Fermentation Process Ethanol and Byproducts The Black Box? Input Materials Anaerobic Microbial Fermentation Z. Lewis Liu
Ethanol Fermentation Process (continued) Z. Lewis Liu glucose 2x pyruvate ADP ATP NAD+ NADH 2 + 2H+ 2x acetaldehyde 2x ethanol 2x CO2 H+ NAD+ regeneration Ethanol Fermentation Process (continued) glycolysis
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
Metabolic Conversion Pathways Furfural Furfural Furfuryl Alcohol HMF (5-Hydroxymethylfurfural) HMF HMF Alcohol? HMF other than HMF Alcohol Z. Lewis Liu
Metabolic Conversion Pathways (cont.) Identification of 2,5-bis-hydroxymethylfuran HMF 2,5-bis-hydroxymethylfuran Z. Lewis Liu
Biotransformation of HMF Metabolic Conversion Pathways (cont.) Biotransformation of HMF Z. Lewis Liu
Adaptive Response Z. Lewis Liu
Potential for Tolerance Improvement Enhanced Detoxification of HMF Enhanced Tolerance Against HMF Z. Lewis Liu
Potential for Tolerance Improvement (cont.) Enhanced Tolerance and Detoxification of Furfural Z. Lewis Liu
IMPROVEMENT POTENTIAL Enhanced Biotransformation Enhanced Tolerance Enhanced Detoxification IMPROVEMENT POTENTIAL Z. Lewis Liu
Byproduct Utilization Biomass Hydrolysates Bring New Ingredients into Fermentation Z. Lewis Liu
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) Cy3 Cy5 Z. Lewis Liu
Tolerance Mechanisms by Functional Genomics Genomic Expression Z. Lewis Liu
Gene expression response to HMF Genomic Expression (cont.) Gene expression response to HMF Initial response Over time responses Gene number Repressed 203 No change Repressed quickly 55 Repressed slowly 30 54 Enhanced 35 Back to neutral 37 15 ∑ 429 Z. Lewis Liu
Genomic Expression (cont.) Repressed-Repressed Enhanced-Enhanced Enhanced-Repressed Z. Lewis Liu
Genomic Expression (cont.) 1 2 3 4 5 6 7 8 9 Z. Lewis Liu
Novel Strain Design and Genetic Engineering Stress Tolerance Mechanisms Biological Experimental Confirmation Hypothesis Development Robust strain Improvement Novel Strain Design and Genetic Engineering Functional Genomics Pathway Analysis Tolerant Strain Resources Gene Cloning In vitro Expression Fermentation Tests Biochemical Pathway Analysis Integrated Programming Novel Strain Design In situ Detoxification Recombinant DNA High Yield / Low Cost Z. Lewis Liu
Biomass Conversion to Ethanol Mission Objectives More Efficient Biomass Conversion to Ethanol Z. Lewis Liu