"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.
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
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
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]
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.
Principal Investigator: Jay Keasling
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
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.
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