genomic.science@science.doe.gov
Office of Biological and Environmental Research
U.S. Department of Energy Office of Science
Susan Gregurick
Office of Biological and Environmental Research
U.S. Department of Energy Office of Science
genomic.science@science.doe.gov
Office of Biological and Environmental Research
U.S. Department of Energy Office of Science
Office of Biological and Environmental Research
U.S. Department of Energy Office of Science
"DOE JBEI is designed to be an engine of ingenuity, dynamically
organized with all the scientific teams working together in a single
location to enable researchers to
share ideas and address cellulosic
biomass problems at a systems
level. Within 60 miles of JBEI, we
have available some of the world's
foremost authorities on energy,
plant biology, systems and synthetic
biology, imaging, nanoscience,
and computation, plus the
highest concentration of national
laboratories and research universities in the nation."
– Jay Keasling
Jay Keasling is the JBEI Chief Executive Officer and a University of California, Berkeley professor of chemical engineering. He also is an award-winning scientific researcher and one of the world's leading authorities on synthetic biology.
Project Description: The DOE Joint BioEnergy Institute (JBEI) is a six-institution partnership led by Lawrence Berkeley National Laboratory (Berkeley Lab). It is based in the San Francisco Bay Area, which is fast becoming a hub of renewable energy research and development, and is headquartered in a new facility in Emeryville, close to its partner institutions. JBEI researchers are engineering microbes and enzymes to process the complex sugars of lignocellulosic biomass into biofuels that can directly replace gasoline. Among the strategies they employ to produce these next-generation biofuels are the tools of synthetic biology. By developing new bioenergy crops, JBEI researchers will improve the fermentable content of biomass and transform lignin into a source of valuable new products.
JBEI's research revolves around four interdependent efforts that focus on (1) developing new bioenergy crops, (2) enhancing biomass deconstruction, (3) producing new biofuels through synthetic biology, and (4) creating technologies that advance biofuel research. Some recent highlights of JBEI research are featured.
Research Strategy:
1. Developing New Bioenergy Crops
To increase our understanding of genes and enzymes
involved in the synthesis and modification of plant cell
walls, JBEI researchers are using well-characterized
genomes and genetic-engineering tools established for
rice and Arabidopsis (a small flowering plant related to
mustard). These two model systems are ideal for research
because their development from seed to mature plant
takes only weeks or months, rather than the year or more
required for energy crops such as switchgrass and poplar.
Genetic insights from rice (a model for grasses) and Arabidopsis
(a model for trees) will accelerate the development
of new energy crops (see figure, Bioenergy Crop Research
at JBEI).
In addition, JBEI scientists are investigating metabolic pathways involved in lignin biosynthesis. The research may lead to development of plants that can be deconstructed more easily. This unique basic research program also could help transform lignin into a valuable source of chemicals and polymers, while improving the economics of converting cellulosic biomass into fuels.
Bioenergy Crop Research at JBEI. JBEI's director of Grass Genetics, Pam Ronald, in the Miscanthus plot at the University of California, Davis. [Photo courtesy of Dan Putnam, UC, Davis]
JBEI Technology Development to Advance Biofuel Research. JBEI's Chris Petzold and Alyssa Redding develop and apply mass spectrometry approaches to sort through the complex protein mixtures in biological cells and detect multiple target proteins in the same sample. [Photo by Dino Vournas, Sandia National Laboratories]
2. Enhancing Biomass Deconstruction
Scientists at JBEI are developing new pretreatment
approaches and enzymes that enhance cellulose conversion
to sugars and minimize the formation of toxic by-products. A
large focus is on the use of ionic liquids, salts that are liquid
rather than crystalline at room or near-room temperatures.
JBEI researchers are investigating both the effects of ionic
liquids on biomass and the recovery of sugars from the liquid
product through the use of solvents. They also are exploring
a broad range of environments, from rainforests to compost,
to discover and isolate new enzymes that more efficiently
degrade cellulose and lignin. JBEI studies of the mechanisms
of biomass deconstruction at the molecular level will enable
new insights and approaches for the efficient conversion of all
plant components to useful products.
3. Producing New Biofuels Through Synthetic
Biology
JBEI researchers are applying synthetic biology techniques
and mathematical models of metabolism and gene regulation
to engineer microorganisms that convert the sugars released
from biomass deconstruction into advanced biofuels, such as
alcohols (e.g., butanol) and alkanes. These next-generation
biofuels will yield almost as much energy per volume as gasoline
and will be transportable through existing fuel pipelines
(see sidebar, Synthetic Biology). Biologically produced
alkanes and other oil-like hydrocarbons could replace gasoline
in today's cars on a gallon-for-gallon basis.
4. Creating Technologies that Advance
Biofuel Research
JBEI scientists are creating new, broadly applicable technologies
to advance research that will speed biofuel development
(see figure, JBEI Technology Development to
Advance Biofuel Research). Among these technologies
is a novel chip-based system that can be used to identify
new enzymes with cellulose- and lignin-degrading activities.
In addition, the researchers are constructing automated
microfluidic platforms that can screen hundreds of enzymatic
reactions simultaneously to help identify the best
enzymes for biomass deconstruction. Technologies also are
being developed for rapid high-resolution imaging to visualize
and characterize the effects of pretreatment protocols
on plant biomass. These and other enabling technologies
are generating large volumes of data that are collected and
catalogued in a centralized database and then analyzed using
new bioinformatic tools.
Synthetic Biology Building Novel Biological Systems for Useful Purposes
Synthetic biologists design and build novel organisms to generate products not made by natural systems. This process may involve constructing entirely new biological systems from a set of standard parts—genes, proteins, and metabolic pathways—or redesigning existing biological systems. The tools of synthetic biology also can be used to study the interior of living cells at the molecular level, providing critical new information and insight into the machinery of life and the natural world. Synthetic biology holds promise for advances in many areas, including the development of renewable, carbon-neutral energy sources; nonpolluting biological routes for the production of chemicals; safer and more effective pharmaceuticals; and better environmental remediation technologies.
At JBEI, researchers are using synthetic biology to develop new platform hosts for producing enzymes and fuels and to create biomolecular parts and devices for constructing new fuel-generating organisms and improved plants. Among other advances, such goals will be achieved through the improved capabilities of fermentative organisms to tolerate processing conditions and inhibit unwanted by-products. Capabilities also will be engineered into fuel-producing organisms to convert 5-carbon sugars into fuel and make use of lignin monomers. Following the strategy that biological systems can be revamped more effectively or built from scratch if standardized parts are employed, investigators are assembling a catalog of well-characterized biosynthetic components to help in designing, testing, optimizing, and implementing integrated large-scale biosynthetic units. These tools and principles, used by JBEI Chief Executive Officer Jay Keasling to develop a relatively inexpensive microbial-based alternative for producing the antimalarial drug artemisinin, will aid in developing the next generation of biofuels.
[Image courtesy of Manfred Auer, Lawrence Berkeley National Laboratory]
Industry Partnerships
To promote the transfer of JBEI inventions to private industry
for commercial development that can benefit the nation,
JBEI has established collaborations with companies that have
relevant scientific and marketing capabilities in energy, agribusiness,
and biotechnology. The JBEI Industry Partnership
Program provides companies with opportunities to contribute
to JBEI and become part of the JBEI community. To
further help ensure that its science ultimately will be able to
serve national needs, JBEI has established an advisory committee,
with representatives from the entire spectrum of the
biofuel industry. For more information on JBEI'S collaborations
with industry, see jbei.org/industry/.
Education and Outreach
Educational efforts at JBEI build on strong undergraduate,
graduate, and postdoctoral training programs, plus
nationally recognized K–12 and community college science
outreach programs already in place at JBEI's member
institutions. In addition to starting a new student fellowship
program, JBEI is collaborating with the University of
California, Berkeley's Management of Technology Program
to enable young scientists and engineers to develop
biofuel-related business plans. JBEI's own education and
outreach programs include internships, scientific academies,
seminars, and collaborations with academic and industrybased
science institutions. In addition to external education
opportunities, JBEI also offers its researchers in-house
seminars as resources for ongoing education.
Lead Institution: Lawrence Berkeley National Laboratory, Berkeley, California
Principal Investigator: Jay Keasling
JBEI Partners:
Location of Center: San Francisco Bay (East), California
Dissolving Cell-Wall Compounds with Ionic Liquids. These confocal fluorescence images show switchgrass cell walls (A) before pretreatment with the EmimAc ionic liquid and (B) 10 minutes after treatment, in which the cell walls have swollen in size, a prelude to complete solubilization of cellulose, hemicellulose, and lignin. [Image courtesy of Seema Singh, Sandia National Laboratories]
New Approach to Visualize Biomass Solubilization
During Ionic Liquid Pretreatment.
JBEI researchers have developed a technique, based on the
natural autofluorescence of plant cell walls, that enables the
dynamic imaging of biomass solubilization during ionic
liquid pretreatment. Using this technique, researchers can
accurately and quickly assess the ionic liquid's performance
without the need for labor-intensive and time-consuming
chemical and immunological labeling. Working with switchgrass
and using the ionic liquid known as 1-n-ethyl-3-methylimidazolium
acetate (EmimAc), the researchers observed
a rapid swelling of secondary plant cell walls (see figure)
within 10 minutes of exposure at relatively mild pretreatment
temperatures (120°C). This reaction indicates a disruption
of hydrogen bonding within cellulose and between cellulose
and lignin. The swelling was followed by complete
dissolution of biomass over 3 hours. By adding water to the
solubilized biomass mixture, cellulose can be precipitated
out and separated from the lignin, which remains in solution.
This recovered cellulose was efficiently hydrolyzed into its
sugar components by a commercial cellulase cocktail over a
relatively short time interval. Currently, those ionic liquids
that are most effective at dissolving plant cell-wall polymers
are prohibitively expensive for use on a mass scale. Understanding
how ionic liquids are able to dissolve lignocellulosic
biomass could pave the way for finding new and better varieties
for use in biofuel production. This research was reported
in Singh, S., B. A. Simmons, and K. P. Vogel. 2009. "Visualization
of Biomass Solubilization and Cellulose Regeneration
During Ionic Liquid Pretreatment of Switchgrass," Biotechnology
and Bioengineering 104(1), 68–75.
Unique Database Provides Functional
and Phylogenomic Information
for Rice Glycosyltransferases.
JBEI researchers have made major advances in comprehensively
identifying all rice glycosyltransferases (GT), an important
class of enzymes involved in synthesizing polysaccharide
sugars in plant cell walls. Because rice and other grasses such
as switchgrass and Miscanthus share similar cell-wall characteristics,
whole genome–scale analysis of rice has enabled
the discovery of several candidate genes for more in-depth
functional analysis that can help researchers understand
and manipulate grass cell walls for biofuel production. This
research has led to the development of JBEI's Rice GT Database,
a publicly available resource for integrating and displaying
diverse sets of functional genomic information for GTs
(ricephylogenomics.ucdavis.edu/cellwalls/gt/). The database
contains information on 793 putative gene models for rice
GTs, and the loci for these genes are distributed across all 12
rice chromosomes. In addition to defining phylogenetic relationships
among groups of rice GT genes based on sequence
similarity, JBEI researchers also compared the number of
different GT gene models identified for rice, Arabidopsis, and
poplar (Populus trichocarpa). From the hundreds of possible
GT genes that have been identified, scientists revealed 33
rice-diverged GTs that are highly expressed in vegetative,
aboveground tissues and that serve as prime targets for mutagenesis
studies and enzyme activity screens. This database
was reported in Cao, P. J., et al. 2008. "Construction of a Rice
Glycosyltransferase Phylogenomic Database and Identification
of Rice-Diverged Glycosyltransferases," Molecular Plant
1(5), 858–77.
Growth on Switchgrass Changes Microbial Community Composition. The populations of microbes present after 31 days of growth on switchgrass (indicated in red) are considerably different from those populations in the compost community (indicated in blue). This suggests a selection and enrichment of specific populations to degrade switchgrass. [Image from Allgaier et al. 2010]
Compost Microbes Adapted to Produce
Switchgrass-Degrading Enzymes.
By incubating switchgrass with a mix of microbes isolated from
compost, JBEI researchers provided the selective pressure
needed to grow a new microbial community enriched with
enzymes that degrade cell-wall polymers specific to switchgrass.
The sample was incubated in a bioreactor for 31 days
under typical composting conditions. Metagenomic sequencing
of the switchgrass-adapted compost (SAC) community on
day 31 was carried out to investigate the sample's diverse pool
of glycoside hydrolases—enzymes that break bonds between
carbohydrate molecules. The sample contained a high proportion
of genes encoding enzymes that attack the branches and
backbone of a major hemicellulose in grass cell walls. Analysis
of the small-subunit ribosomal RNA (rRNA) isolated from
the microbial community revealed dramatic changes in the community profile with more than a 20-fold increase for
some bacterial populations in the SAC (see figure).
Although metagenomic DNA sequence is highly fragmented,
making isolation of full genes from complex communities difficult,
two full-length genes for cellulose-degrading enzymes
were discovered, synthesized, expressed in Escherichia coli,
and tested for enzyme activity. This research was reported in
Allgaier, M., et al. 2010. "Targeted Discovery of Glycoside
Hydrolases from a Switchgrass-Adapted Compost Community,"
PLoS One 5(1), e8812.
Mass SpectrometryBased Protein Detection
Technique Speeds Optimization of Biofuel Protein
Levels in Metabolically Engineered Microbes.
JBEI researchers have developed a mass spectrometry–
based protein detection technique called multiple-reaction
monitoring (MRM) for identifying microbial proteins that
can convert cellulosic sugars into biofuels. With the MRM
technique, researchers can detect multiple target proteins
in the complex protein mixtures of native cells and rapidly
change the specific proteins to be targeted, something not
possible with conventional protein detection technology.
When coupled to liquid chromatography,
MRM analysis
offers high selectivity and sensitivity. It eliminates background
signal and noise even in the most complex protein. mixtures by utilizing two
targeted points—a peptide
mass and a specific fragment
mass generated by mass spectrometry.
Since the entire mass
range is not scanned and only
combinations of peptide and
fragment masses are monitored,
MRM can be used to
detect and quantify up to 10
different proteins in a single
liquid chromatography
separation.
The MRM technique is
a valuable tool for analyzing
enzyme complexes in a variety
of JBEI projects such as the
synthetic protein scaffold work
reported in Dueber, J. E., et al.
2009. "Synthetic Protein Scaffolds
Provide Modular Control
over Metabolic Flux," Nature
Biotechnology 27(8), 753–59.
Key Genes for Biosynthesis of Hydrocarbon
Biofuels Identified in Bacterium Micrococcus luteus.
JBEI researchers have elucidated the genes and a proposed
biochemical pathway for the production of long-chain
alkenes—key chemical components of petroleum-based
gasoline and diesel fuels—in the bacterium Micrococcus
luteus. Building on insights from microbial alkene research
reported 4 decades ago, JBEI researchers hypothesized that
a key mechanism for long-chain alkene biosynthesis would
involve decarboxylation and condensation of fatty acids. By
searching the genome of the alkene-producing bacterium
M. luteus, researchers found three candidate genes with
conserved sequences associated with condensing enzymes.
Expression of these genes in E. coli resulted in long-chain
alkene production, but additional research will be needed to
reveal the specific biochemical role that each of the enzymes
encoded by these genes plays in alkene synthesis. A wide
range of bacteria has been found to contain genes similar to
those that encode M. luteus alkene biosynthesis enzymes,
so researchers will have an opportunity to learn more about
these enzymes by exploring their diversity in nature. This
research was reported in Beller, H. R., E. B. Goh, and J. D.
Keasling. 2010. "Genes Involved in Long-Chain Alkene Biosynthesis
in Micrococcus luteus," Applied and Environmental
Microbiology 76(4), 1212–23.
