Synthetic Biology in the Quest for Renewable Energy

Jay Keasling

Berkeley Center for Synthetic BiologyUniversity of California &

Lawrence Berkeley National LaboratoryBerkeley, CA 94720

The need for renewable energy

• US Energy demands to grow

• Reduction of US CO2 emissions

• Production of clean, cheap energy

1990: 12 TW 2050: 28 TW

Renewable

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.

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%

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

3-D Cellulose Structure

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.

Hemicellulose structure

Cellulose to ethanol

Taken from Demain et al. 2005. Microbiol. Mol. Biol. Rev. 69:124-154.

Cellulose

Hemicellulose

Cellobiose

XyloseXylobiose

C. thermocellum

C. thermosaccharolyticum

Ethanol

Lactate

Acetate

Cellulase

Hemicellulase

60ºC

Cellulosome structure

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

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

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.

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

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’

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

Why do we need synthetic biology?

• Energy production– Production of hydrogen or ethanol– Efficient conversion of waste into energy– Conversion of sunlight into hydrogen

Why now?

• Advances in computing power

• Genomic sequencing• Crystal structures of

proteins• High through-put

technologies• Biological databases• Diverse biological

sampling/collection

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

Building a Super H2 Producer

Complex Polysaccharides

H2

Specialty & CommodityChemicals Ethanol

Building a new chromosome based on genome sequences

Identification of minimal

gene set

Maximizing renewable

resource utilization

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.

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.

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.

Benefits to LBNL

• Establish a new initiative in synthetic biology.

• Establish a new program in hydrogen/ethanol production.

• Utilize large sequence database from JGI.