1. Synthetic Biology in the Quest for Renewable Energy
Jay Keasling
Berkeley Center for Synthetic Biology
University of California &
Lawrence Berkeley National Laboratory
Berkeley, CA 94720

2. The need for renewable energy
US Energy demands to grow
Reduction of US CO2 emissions
Production of clean, cheap energy
Renewable
1990: 12 TW : 28 TW

3. Biomass: a source for renewable energy
About half of the carbonaceous compounds in terrestrial biomass are cellulose.
The net primary production of biomass is estimated to be 60 Gt/yr of carbon in terrestrial and 53 Gt/yr in marine ecosystems.
Almost all of the biomass produced is mineralized again by enzymes which are provided by microorganisms.
Polysaccharide hydrolysis is one of the most important enzymatic processes on earth.

4. Lignocellulose Nearly universal component of biomass
Consists of three types of polymers:
Cellulose
Hemicellulose
Lignin
All three are degraded by bacteria and fungi
Component
Percent Dry Weight
Cellulose
40-60%
Hemicellulose
20-40%
Lignin
10-25%

5. Cellulose
Cellulose is a chemically homogeneous linear polymer of up to 10,000 D-glucose molecules, which are connected by ß-1,4-bonds.
Taken from

6. 3-D Cellulose Structure

7. Hemicellulose
Hemicellulose is a polysaccharide composed of a variety of sugars including xylose, arabinose, mannose.
Hemicellulose that is primarily xylose or arabinose are referred to as xyloglucans or arabinoglucans, respectively.
Hemicellulose molecules are often branched.
Hemicellulose molecules are very hydrophilic.
They become highly hydrated and form gels.

8. Hemicellulose structure

9. C. thermosaccharolyticum
Cellulose to ethanol
Cellulase
Cellulose
Cellobiose
C. thermocellum
Ethanol
Lactate
60ºC
Hemicellulase
Xylose
Xylobiose
Hemicellulose
C. thermosaccharolyticum
Acetate
Taken from Demain et al Microbiol. Mol. Biol. Rev. 69:

10. Cellulosome structure

11. Cellulosome structure
Stable & flexible
Many subunits
Organization promotes synergistic action
Non-catalytic, multipurpose subunit which is the core of cellulosome structure
Scaffoldin - 1,800 amino acids; single Cellulose Binding Domain; Cohesins; anchors cellulosome to cell surface

12. Cellulosome structure
More active against crystalline than amorphous cellulose
Form lengthened corridors between cell & substrate
Cellulose degradation aided by noncellulosomal cellulases & cellulosomes released into environment

13. Problems
Products other than ethanol or hydrogen are produced from cellulose.
Clostridia are difficult to engineer.
Cellulosome is extremely complex making its transplantation to another microbe a significant hurdle.

14. Goal Improve yield of energy-rich molecules from cellulose
Engineer the cellulosome into a genetically tractable microorganism (e.g., Bacillus subtilis)
Develop clostridium genetics to the point that extraneous metabolic reactions can be eliminated

15. Synthetic Biology De novo design of biological entities
Enzymes
Biomaterials
Metabolic pathways
Genetic control systems
Signal transduction pathways
Need the ability to write a ‘blueprint’

16. Why do we need synthetic biology?
Synthesis of drugs or other molecules not found in nature
Designer enzymes
Designer cells with designer enzymes or existing enzymes

17. Why do we need synthetic biology?
Energy production
Production of hydrogen or ethanol
Efficient conversion of waste into energy
Conversion of sunlight into hydrogen

18. Why now? Advances in computing power Genomic sequencing
Crystal structures of proteins
High through-put technologies
Biological databases
Diverse biological sampling/collection

19. Why here?
LBL has played a central role in the development of most of the technologies that will be essential for synthesizing new bacteria.
Synthetic biology will leverage major LBL programs
Joint Genome Institute
Genomes-to-Life
Advanced Light Source
Molecular Foundry
NERSC

20. Building a Super H2 Producer
Specialty & Commodity
Chemicals H2
Ethanol
Identification of minimal gene set
Building a new chromosome based on genome sequences
Maximizing renewable resource utilization
Complex Polysaccharides

21. Specific aims
Determine chromosomal design rules and construct the basic superstructure for an artificial chromosome for our host organism.
Determine the minimal number of genes necessary for a viable, yet robust bacterium.
Determine the components of the cellulose degrading machinery necessary for cellulose utilization.

22. Integration with LBNL Projects
Joint Genome Institute
Cellulose degraders sequenced by JGI and artificial chromosome sequencing.
Genomes to Life
Transcript and protein profiling using GTL facilities.
Molecular Foundry
The cellulose degradation machinery as a model molecular motor.
Synthetic Biology
New initiative at LBNL and UCB.

23. Technical Challenges
Engineering a completely new organism is a daunting task.
The cellulose degrading machinery is an incredibly complicated molecular machine that will require significant characterization in its native host before it can be engineered into a new host.

24. Benefits to LBNL Establish a new initiative in synthetic biology.
Establish a new program in hydrogen/ethanol production.
Utilize large sequence database from JGI.