Synthetic Biology in the Quest for Renewable Energy

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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

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The need for renewable energy

• US Energy demands to grow
• Reduction of US CO2 emissions
• Production of clean, cheap energy

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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.

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Lignocellulose

• Nearly universal component of biomass
• Consists of three types of polymers
• Cellulose
• Hemicellulose
• Lignin
• All three are degraded by bacteria and fungi

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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
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3-D Cellulose Structure
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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.

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Hemicellulose structure
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Cellulose to ethanol
Cellulase
C. thermocellum
Cellulose
Cellobiose
Ethanol
Lactate
60ºC
Hemicellulase
Xylose Xylobiose
C. thermosaccharolyticum
Hemicellulose
Acetate

• Taken from Demain et al. 2005. Microbiol. Mol.
  Biol. Rev. 69124-154.

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Cellulosome structure
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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

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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

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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.

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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

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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

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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

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Why do we need synthetic biology?

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

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Why now?

• Advances in computing power
• Genomic sequencing
• Crystal structures of proteins
• High through-put technologies
• Biological databases
• Diverse biological sampling/collection

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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

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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
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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.

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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.

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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.

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Benefits to LBNL

• Establish a new initiative in synthetic biology.
• Establish a new program in hydrogen/ethanol
  production.
• Utilize large sequence database from JGI.