1. 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

2. 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

3. 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

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

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

6. 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

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
HMF 2,5-bis-hydroxymethylfuran
Z. Lewis Liu

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

11. Adaptive Response
Z. Lewis Liu

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

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

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

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)
Cy3 Cy5
Z. Lewis Liu

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

18. 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

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

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

21. 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

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