Synthetic Biology in the Quest for Renewable Energy

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 Renewable 1990: 12 TW 2050: 28 TW • US Energy demands to grow • Reduction of US CO2 emissions • Production of clean, cheap energy

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

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 http://www.lsbu.ac.uk/water/hycel.html

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. Cellulose to ethanol Taken from Demain et al. 2005. Microbiol. Mol. Biol. Rev.69:124-154. Cellulase C. thermocellum Cellulose Cellobiose Ethanol Lactate 60ºC Hemicellulase Xylose Xylobiose C. thermosaccharolyticum Hemicellulose Acetate

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 H2Producer 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.