# Presentation: 098150

## 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

## The need for renewable energy

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

## 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

## 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**

**Xylose**
**Xylobiose**

***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?

**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**

**H****2**

**Specialty & Commodity**
**Chemicals**

**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.