BER Research Highlights

Search Date: December 13, 2017

43 Records match the search term(s):


December 14, 2015

Optimizing Microbial Bioproduction of Fuels

Identifying factors that contribute to cell-to-cell variability in lipid production.

The Science
Microbial strains engineered to produce a large amount of lipids hold tremendous promise for the production of biofuels and chemicals. A recent study shed light on underlying causes of microbial cell-to-cell variability in lipid production.

The Impact
The findings revealed that conditions within cells and in the surrounding environment interact to contribute to variability in lipid production. The new insights could lead to strategies that optimize the use of engineered microbial strains for the production of important biofuels and chemicals.

Summary
The microbial production of biofuels and chemicals often does not reach the theoretical maximum yield, even for engineered strains, thereby limiting the reliability of large-scale bioprocessing. To understand the limitations, scientists have started to investigate the reasons for phenotypic diversity of cells within a culture. A team of scientists from the University of Idaho, Environmental Molecular Sciences Laboratory (EMSL), and Massachusetts Institute of Technology used advanced microfluidics combined with Epifluorescent and Raman microscopy at EMSL to study differences in the ability of individual cells of low-yield and high-yield strains of the fungus Yarrowia lipolytica to produce lipids. The researchers found lipid production fluctuated sporadically with time in both strains. The researchers labeled this newly discovered phenomenon “bioprocessing noise.” Furthermore, the high-yield fungal strain showed reduced bioprocessing noise in lipid production than the low-yield fungal strain. This finding indicates differences in the activity of key metabolic genes that contribute to bioprocessing noise and thus cellular diversity in lipid production. Moreover, this variability was amplified by environmental factors such as chemical gradients of nutrients or waste products surrounding cells. Taken together, these findings show extracellular and intracellular fluctuations interact to place an upper limit on the reliability of lipid production and total yield of lipids. This research could pave the way for new strategies to improve the reliability and efficiency of using engineered microbial strains for the production of lipids that could then be converted to valuable biofuels or chemicals.

BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324

PI Contacts
Andreas Vasdekis
University of Idaho
andreasv@uidaho.edu

Gregory Stephanopoulos
Massachusetts Institute of Technology
gregstep@mit.edu

Funding
This work was supported by the U.S. Department of Energy’s Office of Science, Office of Biological and Environmental Research, including support of EMSL, a DOE Office of Science user facility; National Institute of General Medical Sciences of the National Institutes of Health; and a Linus Pauling Fellowship from Pacific Northwest National Laboratory.

Publications
Vasdekis, A. E., A. M. Silverman, and G. Stephanopoulos. 2015. “Origins of Cell-to-Cell Bioprocessing Diversity and Implications of the Extracellular Environment Revealed at the Single-Cell Level,” Nature Scientific Reports 5(17689), DOI: 10.1038/srep17689. (Reference link)

Related Links
EMSL article

Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER


December 04, 2015

Characterizing the Structural Basis of Stereospecificity in Enzymatic Cleavage of Lignin Bonds

Understanding how bacteria digest plant lignin informs engineering efforts to extract value from lignin.  

The Science
To determine the structural basis for stereospecificity of bacterial enzymes involved in lignin bond cleavage, researchers solved the crystal structures of the enzymes involved. The detailed structural and biochemical characterization of the lignin degradation pathway members reveals important new aspects of the enzyme mechanisms and determinants of substrate specificity.

The Impact
Lignin is a combinatorial polymer comprised of monoaromatic units and is a potential source of valuable aromatic chemicals. However, its recalcitrance to chemical or biological digestion presents a major obstacle to the production of second-generation biofuels and other valuable bioproducts. These collaborative studies elucidating mechanisms of lignin degradation may enable the development of efficient pathways for converting lignin into components of advanced biofuels and other bioproducts.      

Summary
Lignin’srecalcitrance to chemical or biological digestion presents a major obstacle to the production of second-generation biofuels and valuable coproducts from lignin’s monoaromatic units. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin was reported in the bacterium Sphingobium sp. SYK-6. In a collaborative effort, researchers from the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) and Joint BioEnergy Institute (JBEI) determined the X-ray crystal structures and biochemical characterizations of several glutathione-dependent β-etherases that participate in the cleavage of lignin. Results from these studies reveal important new aspects of the enzyme mechanisms and the determinants of substrate specificity. As β-aryl ether bonds account for 50 percent to 70 percent of all inter-unit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on lignin valorization.

Contacts
(BER PM)

N. Kent Peters, SC-2.32, kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
George N. Phillips, Jr.
Rice University
georgep@rice.edu

Funding
This work was funded by GLBRC and JBEI (DOE Office of Science, Office of Biological and Environmental Research DE-FC02-07ER64494 and DE-AC02-05CH11231, respectively), additional grants from DOE (Office of Science, Office of Basic Energy Sciences, Contract No. DE-AC02-05CH11231 and DE-AC02-06CH11357), grants from the National Institutes of Health (AGM-12006, GM109456, GM098248, P41GM103399, and S10RR027000), the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817), National Cancer Institute (ACB-12002), and University of Wisconsin-Madison.  

Publications
Helmich, K., et al. 2015.  “Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-aryl Ether Bonds in Lignin,” The Journal of Biological Chemistry, DOI: 10.1074/jbc.M115.694307. (Reference link)
Pereira, J. H., et al. 2016. “Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-aryl Ether Cleavage Pathway from Sphingobium sp SYK-6,” The Journal of Biological Chemistry, DOI: 10.1074/jbc.M115.700427. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 27, 2015

Mass Spectrometry Deduces Selectivity of Glycoside Hydrolases for Degrading Biomass Polysaccharides

Improving the annotation of glycoside hydrolases and their phylogenetic trees.

The Science
Multiple classes of polysaccharide-degrading enzymes are used to hydrolyze plant biomass into fermentable sugars for conversion to biofuels. However, there are large numbers of suspected polysaccharide-degrading enzymes whose activities have not been determined biochemically. Researchers have now determined the reaction specificity and other parameters for several of these uncharacterized enzymes using a special mass spectroscopy system along with artificial substrates.

The Impact
Improving the annotation of glycoside hydrolase (GH) phylogenetic trees will improve understanding of the function, synergy, and stability of these enzymes and thereby the creation of biomass-degrading enzymatic cocktails.  

Summary
Researchers at the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) have used chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides to study the reactivity of several enzymes representative of GH function. Patterns of reactivity identified with these NIMS probes provide a diagnostic approach to assess reaction selectivity as well as comparative apparent rate information. Their results show diagnostic patterns for reactions of a β-glucosidase, relaxed but varied specificity of several endoglucanases, and high specificity of a cellobiohydrolase with the model substrate. The researchers also modeled time-dependent reactions of these enzymes by numerical integration, providing a quantitative basis to make functional distinctions among reactive properties, thus providing a new approach to enhance the annotation of GH phylogenetic trees with functional measurements. This research was carried out in collaboration with researchers at DOE’s Joint BioEnergy Institute (JBEI).

Contacts (BER PM)
N. Kent Peters, SC-23.2, kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
Brian Fox
University of Wisconsin-Madison
bgfox@biochem.wisc.edu

Funding
GLBRC and JBEI are supported by DOE’s Office of Science, Office of Biological and Environmental Research through contracts DE-FC02-07ER64494 and DE-AC02-05CH11231, respectively.

Publications
Deng, K., T. E. Takasuka, C. M. Bianchetti, L. F. Bergeman, P. D. Adams, T. R. Northen, and B. G. Fox. 2015. “Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases,” Frontiers in Bioengineering and Biotechnology 3(165), DOI: 10.3389/fbioe.2015.00165. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 13, 2015

Cyanobacterial Alkanes: Today’s Bacterial Antifreeze, Tomorrow’s Fuel

A promising biofuel molecule helps the photosynthetic bacteria that naturally produce it tolerate cold temperatures.

The Science
Cyanobacteria, photosynthetic microorganisms, contain a unique and universal pathway that converts fatty acids to alkanes, a promising biofuel candidate. Recent spectroscopic and modeling studies illuminated how the produced alkanes allow cyanobacteria to adjust photosynthetic activity to tolerate cold temperatures.

The Impact
Understanding the natural function of alkanes in cyanobacteria may lead to production of these molecules as biofuels. These bacteria-produced alkanes are excellent fuel candidates because they have high-energy content and are highly compatible with existing infrastructure for petroleum-based fuel distribution and use.

Summary
Cyanobacteria are photosynthetic bacteria that, like plants, consume carbon dioxide and produce oxygen through photosynthesis. All cyanobacterial membranes contain diesel-range C15-C19 hydrocarbons in high concentration and the production pathways for these metabolites are exclusive to cyanobacteria. In this study, the model cyanobacterium, Synechocystis sp. PCC 6803, was modified to produce no alkanes, and the resulting strain grew poorly at low temperatures. To understand the growth defect, the researchers assessed the redox kinetics of how cyanobacteria convert solar energy into chemical energy in the form of adenosine triphosphate (ATP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). ATP and NADPH are produced using a linear and a cyclic pathway with the pigment-protein complex, photosystem I (PSI), as the hub to both. The modified strain made greater use of the cyclic pathway, which raises the ATP:NADPH ratio, especially at low temperature. This use helps to balance reductant requirements and maintain the redox poise of the electron transport chain. While previous theories held that the cyclic pathway was used in a fixed ratio to the linear pathway, the researchers demonstrated that the cyclic pathway responds dynamically to the environment and that alkanes play a role in this response. Flux balance computational analysis showed that an intermediate use of the cyclic pathway (circa one-fourth that of the linear pathway) maximized growth as well. From this analysis, the team concluded that the lack of membrane alkanes required greater use of the cyclic pathway, presumably to maintain redox poise. In turn, such an increase compromises growth by activating energy-inefficient pathways. This study highlights the unique and universal role of medium-chain hydrocarbons in cyanobacteria: they regulate redox balance and reductant partitioning in these photosynthetic cells under stress.

Contacts
(BER PM)
Dawn Adin, SC-23.2, dawn.adin@science.doe.gov, 301-903-0570

(PI Contact)
Himadri B. Pakrasi  
Washington University in St. Louis
pakrasi@wustl.edu

Funding
This work was funded by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Biological Systems Science Division, Genomic Science Program.

Publications
Berla, B., R. Saha, C. Maranas, and H. Pakrasi. 2015. “Cyanobacterial Alkanes Modulate Photosynthetic Electron Flow to Assist Growth under Cold Stress,” Scientific Reports 5, 14894. DOI: 10.1038/srep14894. (Reference link)

Related Links
Pakrasi Laboratory Website
Maranas Laboratory Website

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Cyanobacteria produce alkanes that modulate the balance between different energy production pathways. This feature allows these photosynthetic bacteria to thrive across a range of environmental temperatures.

Image modified from Berla, B., et al. 2015. “Cyanobacterial Alkanes Modulate Photosynthetic Electron Flow to Assist Growth under Cold Stress,” Scientific Reports 5, 14894. DOI: 10.1038/srep14894.



September 22, 2015

Identifying Specific Preferences in Organic Compound Consumption by Desert Soil Microbes

Every natural soil ecosystem hosts a great diversity of microbes that consume complex organic matter and transform it to simpler small carbon compounds (metabolites) or gaseous endproducts such as carbon dioxide. This decompositional microbial activity transforms organic compounds in the soil, playing a critical role in the global carbon cycle. To determine the functional characteristics of a microbial community’s different members, it is necessary to understand the complex mixture of metabolites present in their environment and to determine which compounds are preferentially consumed by each microorganism. Researchers at Lawrence Berkeley National Laboratory and collaborating institutions have used new exometabolomics techniques to quantitatively analyze the compounds consumed by seven bacterial species isolated from soil crusts in the desert environment of the Colorado Plateau. In these arid environments, most of the organic matter is produced by photosynthetic bacteria and released in the form of metabolites that other microbes can consume and further transform. The investigators discovered that each of the seven species consumes only 13% to 26% of the nearly 500 metabolites produced by these bacteria, and only 0.4% of the metabolites are used by all of them. These different feeding habits may represent a form of ecological niche specialization and may play important roles in maintaining non-overlapping diversity within microbial consortia. This study constitutes a significant advance in our understanding of how microbes in terrestrial ecosystems transform soil organic matter and may affect atmospheric carbon dioxide levels.

Reference: Baran, R., E. Brodie, J. Mayberry-Lewis, E. Hummel, U. N. Da Rocha, R. Chakraborty, B. Bowen, U. Karaoz, H. Cadillo-Quiroz, F. Garcia-Pichel, and T. Northen. 2015. “Exometabolite Niche Partitioning Among Sympatric Soil Bacteria,” Nature Communications 6(8289), DOI:10.1038/ncomms9289. (Reference link)

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 21, 2015

Neutron Crystallography Visualizes How Nature’s Most Efficient Enzyme Works

Enzymes play a critical role in all aspects of life by speeding up specific chemical reactions in living cells. The glycoside hydrolases (GHs) are a group of enzymes that catalyze the breakdown of large quantities of organic matter in nature, specifically cellulose and hemicellulose, and that are being applied industrially to the conversion of biomass to useful products. GHs speed up the cleavage of an otherwise very stable chemical bond through a complex process that is not well understood. New research led by scientists at Oak Ridge National Laboratory (ORNL) on the key steps in the action of xylanase, a GH that cuts xylan chains in hemicellulose (a major component of biomass) into smaller units, has shown how this enzyme coordinates the movement of hydrogen ions to speed up the breakdown process. The scientists combined information from several neutron and X-ray crystallography experiments to visualize the exact atomic structure of the xylanase during the initial steps of the reaction. They found that a side chain of the enzyme amino acid residue that is key to its activity moves between two orientations to first accept a hydrogen ion and then deliver it to the place where the xylan is to be cut. In the former orientation, the side chain is more basic and thus is able to grab a hydrogen ion from water, whereas in the latter it becomes more acidic and ready to initiate the catalytic process. This publication is the first from the new Macromolecular Neutron Diffractometer (MaNDi) at ORNL’s Spallation Neutron Source. Scientists at Los Alamos National Laboratory, Argonne National Laboratory, the University of Toledo, and universities and user facilities in the People’s Republic of China, Sweden, and Germany collaborated in the research.

Reference: Wan, Q., et al. 2015. “Direct Determination of Protonation States and Visualization of Hydrogen Bonding in a Glycoside Hydrolase with Neutron Crystallography,” Proceedings of the National Academy of Sciences (USA) 112(40), 12,384–389. DOI: 10.1073/pnas.1504986112. (Reference link)

Contact: Roland F. Hirsch, SC-23.2, (301) 903-9009
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 16, 2015

Novel Biological Wiring System Detected in a Methane-Consuming Microbial Symbiosis

Every year, large amounts of methane (CH4) are produced in coastal wetlands and deep ocean sediments through the decay of organic material or seepage from geological reservoirs. Fortunately, microbes consume the majority of this potent greenhouse gas before it reaches Earth’s atmosphere. Although these subsurface environments are typically depleted of oxygen, methane can still be oxidized by symbiotic partnerships between methane-consuming archaea and sulfate-reducing bacteria that collaboratively transfer electrons from methane to sulfate (rather than O2) to generate useful energy. Observed near sites of environmental CH4 production, consortia of cells performing anaerobic oxidation of methane (AOM) form mixed balls composed of tens to hundreds of cells, but the exact mechanism by which they consume CH4 and share energy is not fully understood. In a new study, scientists at the California Institute of Technology used high-resolution microscopy paired with mass spectrometry (NanoSIMS) to examine the relationship between spatial distribution of microbes and metabolic processes in AOM consortia. To their surprise, the researchers found that metabolically active partner microbes did not need to be closely associated with each other, even though each organism performs only half of the critical methane-consuming reaction. Using data from these studies, the team constructed a computational model of consortial metabolism that predicted an extracellular conduit allowing direct transfer of electrons between the organisms. By re-examining the genomes of both microbes, the team identified a previously overlooked set of genes in the archaeal partner encoding an electron transfer system similar to those observed in known electroconductive bacteria. Histological staining was then used to detect this system in active AOM consortia, revealing components arrayed across the extracellular space between the microbes. These results indicate the presence of a biological wiring system within AOM consortia that allows the two partners to more efficiently consume methane, share resulting energy, and form larger consortial structures than would otherwise be possible. These findings reveal another new aspect of the diverse metabolic capacities present in the microbial world and considerably advance our understanding of a key microscale mechanism driving a carbon cycle process of global significance.

Reference: McGlynn, S. E., G. L. Chadwick, C. P. Kempes, and V. J. Orphan. 2015. “Single Cell Activity Reveals Direct Electron Transfer in Methanotrophic Consortia,” Nature, DOI: 10.1038/nature15512. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


September 12, 2015

Elimination of Non-Productive Fermentation Products Boosts Cellulosic Ethanol Production in Consolidated Bioprocessing

Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. In addition to ethanol, however, C. thermocellum produces a number of unwanted fermentation products such as organic acids and gaseous hydrogen, which divert energy and carbon from the desired fermentation product, ethanol. Researchers at the Department of Energy’s BioEnergy Science Center sought to eliminate these non-target fermentation products in order to increase ethanol yields. In doing so, they created C. thermocellum strain AG553 by deleting genes involved in the production of acetate, formate, lactate, and hydrogen gas. Strain AG553 showed a two- to three-fold increase in ethanol yield relative to the wild type on all substrates tested. When grown in a defined medium with 5 g/L of soluble disaccharide cellobiose as the carbon source, the mutant strain produced greater than two-fold more ethanol than the wild type strain. It exceeded 70% of theoretical ethanol yield with no appreciable amounts of other fermentation products detected and H2 production reduced five-fold. Wild type C. thermocellum will naturally acidify a non-buffered medium during fer­mentation by production of organic acids and limit ethanol production by limiting growth. The elimination of organic acid production suggested that strain AG553 might be capable of growth under higher substrate loadings in the absence of pH control. The maximum titer of wild type C. thermocellum was only 14.1 mM ethanol on 10 g/L Avicel. For strain AG553, final ethanol titer peaked at 73.4 mM in on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to carbon dioxide accumulation. With the elimination of the non-target fermentation metabolic pathways, AG553 is the best ethanol-yielding CBP strain to date. It will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass into advanced biofuels other than ethanol.

Reference: Papanek, B., R. Biswas, T. Rydzak, and A. M. Guss. 2015. “Elimination of Metabolic Pathways to All Traditional Fermentation Products Increases Ethanol Yields in Clostridium thermocellum,”Metabolic Engineering, DOI: 10.10/16/j.ymben.2015.09.002. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 27, 2015

Eucalyptus Trees with Reduced Lignin Content Display Reduced Recalcitrance

Lignocellulosic materials offer an attractive replacement for food-based crops used to produce ethanol, but understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of lignocellulosic biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. Changes to lignin gene expression in switchgrass and Populus have shown increased sugar release and reduced recalcitrance. Researchers at the Department of Energy’s BioEnergy Science Center have sought to transfer these results to eucalyptus, a fast-growing, warm climate, woody biofeedstock also suitable for cellulosic biofuel production. The researchers genetically engineered reduced gene expression of two key lignin biosynthesis enzymes, cinnamate 4-hydroxylase (C4H) and p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H), in eucalyptus. The engineered plants were evaluated for cell wall composition and reduced recalcitrance. Eucalyptus trees with down-regulated C4H or C3'H expression displayed lowered overall lignin content than the control samples. The C3'H and C4H down-regulated lines also had different lignin compositions when compared to the control eucalyptus trees. Both the C4H and C3'H down-regulated lines had reduced recalcitrance as indicated by increased sugar release, which was determined using enzymatic conversion assays utilizing both no pretreatment and a hot water pretreatment. Lowering lignin content rather than altering lignin content was found to have the largest impact on reducing recalcitrance of the transgenic eucalyptus variants. The development of lower recalcitrance trees opens up the possibility of using alternative pretreatment strategies in biomass conversion processes that can reduce processing costs.

Reference: Sykes, R. W., E. L. Gjersing, K. Foutz, W. H. Rottmann, S. A. Kuhn, C. E. Foster, A. Ziebell, G. B. Turner, S. R. Decker, M. A. W. Hinchee, and M. F. Davis. 2015. “Down Regulation of P-Coumaroyl Quinate/Shikimate 3'-Hydroxylase (C3'H) and Cinnamate 4-Hydroxylase (C4H) Genes in the Lignin Biosynthetic Pathway of Eucalyptus urophylla x E. grandis Leads to Improved Sugar Release,” Biotechnology for Biofuels 8,128. DOI: 10.1186/s13068-015-0316-x. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 27, 2015

Structural Characterization of Isolated Poplar and Switchgrass Lignins During Dilute Acid Treatment

A key step in converting cellulosic biomass into sustainable fuels and chemicals is thermochemical pretreatment to reduce plant cell wall recalcitrance. An improved understanding of the chemistry of lignin as it undergoes this processing is critical to the development of renewable biofuel production. Researchers at the Department of Energy’s BioEnergy Science Center (BESC) have studied the behavior of lignin during dilute acid pretreatment (DAP). They isolated lignin from poplar and switchgrass using a cellulolytic enzyme system and then treated it under DAP conditions. Results highlighted that lignin is subjected to depolymerization reactions within the first 2 minutes of DAP, and these changes are accompanied by increased generation of aliphatic and phenolic hydroxyl groups of lignin. These developments are followed by a competing set of depolymerization and repolymerization reactions that lead to a decrease in the content of guaiacyl lignin units and an increase in condensed lignin units as the reaction residence time is extended beyond 5 minutes. A detailed comparison of changes in functional groups and molecular weights of cellulolytic enzyme lignins demonstrated that several structural parameters related to lignin’s recalcitrant properties are altered during DAP conditions. This deeper understanding of the chemical structure of lignin as it undergoes processing is an important step toward the goal of efficient conversion of lignocellulose into renewable biofuel products.

Reference: Sun, Q., Y. Pu, X. Meng, T. Wells, and A. J. Ragauskas. 2015. “Structural Transformation of Isolated Poplar and Switchgrass Lignins During Dilute Acid Treatment,” ACS Sustainable Chemistry and Engineering 3(9), 2203-10. DOI: 10.1021/acssuschemeng.5b00426. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 13, 2015

Enhancing a microbe’s cellulolytic ability for biomass deconstruction

The in vitro activity of the Caldicellulosiruptor bescii secretome to digest lignocellulosic biomass was significantly increased with the addition of the E1 endoglucanase from Acidothermus cellulolyticus.

The Science
The most effective commercial enzyme cocktails currently used to deconstruct biomass in vitro are derived from fungal cellulase components. These fungal cellulases consist of cellobiohydrolases, endoglucanases, and β-D-glucosidases that act synergistically to release sugars from biomass for microbial conversion to products. However, these fungal cellulase components contribute significantly to overall deconstruction costs. As a potentially cost-effective alternative, C. bescii, a cellulolytic thermophile, is a prime candidate for effective consolidated bioprocessing as it contains more than 50 glycoside hydrolases including CelA, a multidomain enzyme. C. bescii’s ability to solubilize lignocellulose could be enhanced with engineering to include an endonuclease with additional activities such as E1 fromA. cellulolyticus, another cellulolytic thermophile.

The Impact
This work provides an understanding of the action and limitations of the CelA enzyme and demonstrates that CelA can act synergistically with the E1 protein to digest cellulose. These results contribute to the knowledgebase that enables enzyme engineering to generate novel enzyme mixtures for biomass deconstruction. The new information could lead to a more economical means of converting biomass to simple sugars for bioproducts production.

Summary
The most effective commercial enzyme cocktails of carbohydrate-active enzymes (CAZymes) used in vitro to pretreat biomass are derived from fungal cellulases. These cellobiohydrolases, endoglucanases, and β-d-glucosidases act synergistically to release sugars for microbial conversion. The genome of the thermophilic bacterium C. bescii encodes a potent set of CAZymes, found primarily as multidomain enzymes. This set of CAZymes exhibits high cellulolytic and hemicellulolytic activity on and allows utilization of a broad range of substrates, including plant biomass, without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in a single protein. E1 is an endo-1,4-β-glucanase from A. cellulolyticus linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates and has been shown in vitro to work synergistically with CelA. To test if the addition of E1 to the C. bescii secretome would improve its cellulolytic activity, U.S. Department of Energy (DOE) BioEnergy Science Center (BESC) scientists cloned and expressed the E1 gene in C. bescii under the transcriptional control of the C. bescii S-layer promoter, and secretion was directed by the addition of the C. bescii CelA signal peptide sequence. Increased activity of the secretome of the strain containing E1 was observed on both carboxymethylcellulose (CMC) and Avicel. Activity against CMC increased on average 10.8 percent at 65 °C, and 12.6 percent at 75 °C. Activity against Avicel increased on average 17.5 percent at 65 °C and 16.4 percent at 75 °C. Thus, expression and secretion of E1 in C. bescii enhanced the cellulolytic ability of its secretome in agreement with in vitro evidence that E1 acts synergistically with CelA to digest cellulose. This result offers the possibility of engineering additional enzymes for improved biomass deconstruction into C. bescii effectively.

Contacts (BER PM)
N. Kent Peters, SC-23.2, kent.peters@science.doe.gov, 301-903-5549

PI Contact
Janet Westpheling
Department of Genetics, University of Georgia, Athens, GA, and BESC, Oak Ridge National Laboratory, Oak   Ridge, TN
janwest@uga.edu

Funding
This research was supported as a subcontract by BESC, a DOE Bioenergy Research Center funded by the Office of Biological and Environmental Research within DOE’s Office of Science (DE-AC05-000R22725).

Publication
Chung, D., J. Young, M. Cha, R. Brunecky, Y. J. Bomble, M. E. Himmel, and J.Westpheling. 2015. “Expression of the Acidothermus cellulolyticus E1 Endoglucanase in Caldicellulosiruptor bescii Enhances Its Ability to Deconstruct Crystalline Cellulose,” Biotechnology for Biofuels 8:113. DOI: 10.1186/s13068-015-0296-x. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 13, 2015

Expression of Heterologous Endoglucanases in Caldicellulosiruptor bescii Enhances Secretome Activity

Currently, the most effective commercial enzyme cocktails of carbohydrate-active enzymes (CAZymes) used in vitro to pretreat biomass are derived from fungal cellulases. These cellobiohydrolases, endoglucanases, and β-d-glucosidases act synergistically to release sugars for microbial conversion. The genome of the thermophilic bacterium, Caldicellulosiruptor bescii, encodes a potent set of CAZymes, found primarily as multidomain enzymes. This set of CAZymes exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in a single protein. E1 is an endo-1,4-β-glucanase from Acidothermus cellulolyticus linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates and has been shown in vitro to work synergistically with CelA. To test if the addition of E1 to the C. bescii secretome would improve its cellulolytic activity, the E1 gene was cloned and expressed in C. bescii under the transcriptional control of the C. bescii S-layer promoter, and secretion was directed by the addition of the C. bescii CelA signal peptide sequence. Increased activity of the secretome of the strain containing E1 was observed on both carboxymethylcellulose (CMC) and Avicel. Activity against CMC increased on average 10.8 % at 65 °C and 12.6 % at 75 °C. Activity against Avicel increased on average 17.5 % at 65 °C and 16.4 % at 75 °C. Thus, expression and secretion of E1 in C. bescii enhanced the cellulolytic ability of its secretome in agreement with in vitro evidence that E1 acts synergistically with CelA to digest cellulose. This result offers the possibility of effectively engineering additional enzymes for improved biomass deconstruction into C. bescii.

Reference: Chung, D., J. Young, M. Cha, R. Brunecky, Y. J. Bomble, M. E. Himmel, and J. Westpheling. 2015. “Expression of the Acidothermus cellulolyticus E1 Endoglucanase in Caldicellulosiruptor bescii Enhances Its Ability to Deconstruct Crystalline Cellulose,” Biotechnology for Biofuels 8, 113. DOI: 10.1186/s13068-015-0296-x. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 10, 2015

Hybrid Spectroscopy Helps Elucidate Fine Cell Wall Structure

A key obstacle to large-scale production of biofuels is the resistance of biomass to deconstruction into simple biomolecules that can be converted to the desired fuels. This so-called recalcitrance is being studied intensively at the cellular level. Non-destructive, simultaneous chemical and physical characterization of materials at the nanoscale is a highly sought-after capability for understanding the underlying mechanisms of this cell wall recalcitrance to deconstruction. However, a combination of physical limitations of existing nanoscale technologies has made achieving this goal challenging. To overcome these obstacles, researchers at the Department of Energy’s BioEnergy Science Center (BESC) have developed a hybrid approach for nanoscale material characterization based on nanomechanical force microscopy in conjunction with infrared photoacoustic spectroscopy. The researchers targeted the outstanding problem of spatially and spectrally resolving plant cell walls. Nanoscale characterization of plant cell walls and the effect of complex phenotype treatments on biomass are challenging but necessary in the search for sustainable and renewable bioenergy. The BESC scientists were able to reveal both the morphological and compositional substructures of the cell walls. They found that the measured biomolecular traits are in agreement with the lower-resolution chemical maps obtained with infrared and confocal Raman microspectroscopies of the same samples. These results should prove relevant in fields such as energy production and storage, as well as medical research, where morphological, chemical, and subsurface studies of nanocomposites, nanoparticle uptake by cells, and nanoscale quality control are in demand.

Reference: Tetard, L., A. Passian, R. H. Farahi, T. Thundat, and B. H. Davison. 2015 “Opto-Nanomechanical Spectroscopic Material Characterization,” Nature Nanotechnology, DOI: 10.1038/NNANO.2015.168. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 22, 2015

Engineered Furfural Tolerance in Caldicellulosiruptor bescii, a Consolidated Bioprocessing Thermophile

Harsh pretreatments are often used to make lignocellulose sugars more accessible for conversion to biofuels. These pretreatments can cause problems for subsequent stages of biofuel production. For example, dilute-acid pretreatment of lignocellulosic biomass creates potent inhibitors of microbial growth and fermentation such as furfural and 5-hydroxymethyl-furfural (5-HMF). The enzymatic reduction of these furan aldehydes to their corresponding less toxic alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenic Escherichia coli. However, this approach has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP), such as Caldicellulosiruptor bescii. To test if C. bescii could be engineered to be more tolerant of these inhibitors, researchers from the Department of Energy’s BioEnergy Science Center (BESC) constructed a strain of C. bescii using a butanol dehydrogenase encoding gene from Thermoanaerobacter pseudethanolicus 39E (BdhA), which had previously been shown to have furfural and 5-HMF reducing capabilities. Heterologous expression of the NADPH-dependent BdhA enzyme conferred increased resistance of the engineered strain to both furfural and 5-HMF relative to the wild-type and parental strains. Further, when challenged with 15 mM concentrations of either furan aldehyde, the ability to eliminate furfural or 5-HMF from the culture medium was significantly improved in the engineered strain. This study represents the first example of engineering furan aldehyde resistance into a CBP-relevant thermophile and further validates C. bescii as being a genetically tractable microbe of importance for lignocellulosic biofuel production.

Reference: Chung, D., T. J. Verbeke, K. L. Cross, J. Westpheling, and J. G. Elkins. 2015. “Expression of a Heat-Stable NADPH-Dependent Alcohol Dehydrogenase in Caldicellulosiruptor bescii Results in Furan Aldehyde Detoxification,” Biotechnology for Biofuels 8,102. DOI: 10.1186/s13068-015-0287-y. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 10, 2015

Consolidated Bioprocessing of Cellulose to an Advanced Biofuel Using a Cellulolytic Thermophile

Consolidated bioprocessing (CBP) has the potential to reduce biofuel and biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously, without the addition of premanufactured cellulases and other hydrolytic enzymes. In particular, Clostridium thermocellum is a promising thermophilic CBP host because of its high cellulose decomposition rate. Toward this end, researchers at the Department of Energy’s BioEnergy Science Center (BESC) researchers engineered C. thermocellum to produce isobutanol, an advanced biofuel. Metabolic engineering for isobutanol production in C. thermocellum is hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. Engineering of the isobutanol pathway into C. thermocellum was facilitated by first cloning plasmids into Escherichia coli before transforming these constructs into C. thermocellum for testing and optimization. Among these engineered strains, the best isobutanol producer was selected. Interestingly, both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (KIVD) were expressed and found to be responsible for isobutanol production. A single crossover integration of the plasmid into the chromosome resulted in a stable strain not requiring antibiotic selection. This strain produced 5.4 g/L of isobutanol from cellulose in minimal medium at 50°C within 75 hours, corresponding to 41% of theoretical yield. While there is significant room for further optimization, this initial engineering of a cellulolytic thermophile to produce an advanced biofuel demonstrates the potential of this strategy to help create a sustainable and commercially viable biofuel.

Reference: Lin, P. P., L. Mi, A. H. Morioka, K. M. Yoshino, S. Konishi , S. C. Xu , B. A. Papanek, L. A. Riley, A. M. Guss, and J. C. Liao. 2015. “Consolidated Bioprocessing of Cellulose to Isobutanol Using Clostridium thermocellum,” Metabolic Engineering 31, 44-52. DOI:10.1016/j.ymben.2015.07.001. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 04, 2015

Engineering Restricted Lignin and Enhanced Sugar Deposition in Secondary Cell Walls Enhances Monomeric Sugar Release

Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. However, the lignin component, a complex and interlinked phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis to convert them to sugars. Therefore, before enzymatic hydrolysis, biomass must first be pretreated to make it more susceptible to saccharification and release high yields of fermentable sugars. To reduce the impact of lignin on limiting saccharification, researchers at the Department of Energy’s Joint BioEnergy Institute (JBEI) engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the plant’s vessels, and polysaccharide deposition was enhanced in fiber cells. Growth of these engineered plants showed little to no apparent negative impact on growth phenotype. Analyses of these engineered Arabidopsis plants were conducted to determine if the engineered plants would yield more sugars than wild type. Both wild type and engineered plant biomasses were treated with an ionic liquid at either 70°C for 5 hours or 140°C for 3 hours. After pretreatment at 140°C and subsequent saccharification, the relative peak sugar recovery from biomass of engineered plants and wild type was not statistically different. However, reducing the pretreatment temperature to 70°C resulted in a higher peak sugar recovery for the engineered lines, but a significant reduction in the peak sugar recovery obtained from the wild type. These results demonstrate that employing cell wall engineering to decrease the recalcitrance of lignocellulosic biomass has the potential to drastically reduce the energy required for effective pretreatment.

Reference: Scullin, C., A. G. Cruz, Y.-D. Chuang, B. A. Simmons, D. Loque, and S. Singh. 2015. “Restricting Lignin and Enhancing Sugar Deposition in Secondary Cell Walls Enhances Monomeric Sugar Release After Low Temperature Ionic Liquid Pretreatment,” Biotechnology for Biofuels 8, 95. DOI: 10.1186/s13068-015-0275-2. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 29, 2015

Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by Rhodopseudomonas palustris

A major barrier to efficient conversion of lignocellulosic materials to biofuels is the sensitivity of microbes to inhibitory compounds formed during biomass pretreatment. Aromatics derived from lignocellulose are a major class of inhibitors that typically are not metabolized by microbes commonly used as biocatalysts. However, the purple nonsulfur bacterium Rhodopseudomonas palustris is known to utilize aromatic compounds such as benzoate or p-hydroxybenzoate under anaerobic conditions. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center (GLBRC) have now shown that R. palustris is able to remove a majority of the aromatic compounds present in corn stover hydrolysates while leaving the sugars intact. The conditioned hydrolysate supported improved growth of a second microbe that was not able to grow in untreated hydrolysate. GLBRC researchers also found that most of the aromatic compounds were metabolized via the known R. palustris benzoyl-coenzyme A (CoA) pathway. Furthermore, the use of benzoyl-CoA pathway mutants prevents complete degradation of the aromatics and allows for production of selected products that may be recovered as coproducts from fermentations. This work presents the first demonstration of a microbe’s ability to metabolize and remove mixed aromatics in biomass hydrolysate, compounds that are detrimental to most microbes and generally unsuitable as carbon sources. This knowledge may inform the design of new microbes for bioconversion that can generate valuable coproducts from fermentation of sugars in lignocellulosic biomass.

Reference: Austin, S., W. S. Kontur, A. Ulbrich, J. Z. Oshlag, W. Zhang, A. Higbee, Y. Zhang, J. J. Coon, D. B. Hodge, T. J. Donohue, and D. R. Noguera. 2015. “Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by Rhodopseudomonas palustris,” Environmental Science and Technology 49(14), 8914–22. DOI: 10.1021/acs.est.5b02062. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 17, 2015

Long-Term Study Alleviates Water-Use Concern for Biofuel Crops

Potential water requirements are a significant concern for large-scale production of biofuel crops. Studying water use for plant communities across years of varying water availability can indicate how terrestrial water balances will respond to climate change and variability as well as to land cover change. Perennial biofuel crops, likely grown mainly on marginal lands of limited water availability, provide an example of a potentially extensive future land-cover conversion. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center measured growing-season evapotranspiration based on daily changes in soil profile water contents in five perennial systems—switchgrass, Miscanthus, native grasses, restored prairie, and hybrid poplar—and in annual maize (corn) in a temperate humid climate (Michigan, USA). Three study years (2010, 2011, and 2013) had normal growing-season rainfall, whereas 2012 was a drought year with about half to a third normal rainfall. Overall growing-season mean evapotranspiration for the four years did not vary significantly among corn and the perennial systems. Differences in biomass production largely determined variation in water-use efficiency. Miscanthus had the highest water-use efficiency in both normal and drought years, followed by maize; the native grasses and prairie were lower and poplar was intermediate. Measured water use by perennial systems was similar to maize across normal and drought years and contrasts with earlier modeling studies suggesting that rain-fed perennial biomass crops in this climate have little impact on landscape water balances, whether replacing rain-fed maize on arable lands or successional vegetation on marginal lands. Results also suggest that crop evapotranspiration rates, and thus groundwater recharge, streamflow, and lake levels, may be less sensitive to climate change than has been assumed.

Reference: Hamilton, S. K., M. Z. Hussain, A. K. Bhardwaj, B. Basso, and G. P. Robertson. 2015. “Comparative Water Use by Maize, Perennial Crops, Restored Prairie, and Poplar Trees in the U.S. Midwest,” Environmental Research Letters 10, 064015. DOI:10.1088/1748-9326/10/6/064015. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 15, 2015

New Microfluidics DNA Assembly Platform

Microbes are being engineered for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful for this process, further improvements are needed to lower the barriers scientists face if they plan to enter this growing field. Researchers at the Department of Energy’s Joint BioEnergy Institute have developed an innovative microfluidic platform for assembling DNA fragments, a critical step in the entire process. The new system uses volumes 10 times lower than current microfluidic platforms and has integrated region-specific temperature control and on-chip transformation. Integration of these steps in a single device minimizes the loss of reagents and products compared to conventional methods, which require, for example, multiple pipetting steps. For assembling DNA fragments, researchers implemented three commonly used DNA assembly protocols on the new microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). Assembly of two combinatorial libraries of 16 plasmids each demonstrated the utility of these microfluidic methods. Each DNA plasmid was transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. This platform likely will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids, helping to expedite the overall synthetic biology process for biofuels development.

Reference: Shih, S. C. C., G. Goyal, P. W. Kim, N. Koutsoubelis, J. D. Keasling, P. D. Adams, N. J. Hillson, and A. K. Singh. 2015. “A Versatile Microfluidic Device for Automating Synthetic Biology,” ACS Synthetic Biology, DOI: 10.1021/acssynbio.5b00062. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


June 12, 2015

Phenolic Amides are Potent Inhibitors of de novo Nucleotide Biosynthesis

Lignocellulose-derived hydrolysates contain several different inhibitors (collectively called lignotoxins or LTs) that arise during pretreatment of biomass. Determining the mechanisms by which yeast or bacteria are adversely affected by LTs is a key step toward improving the efficiency of fermentation and bioconversion. Prior work has established that LTs present in ammonia pretreated corn stover hydrolysates inhibit growth and sugar utilization in Escherichia coli. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center (GLBRC) have now keyed in on two phenolic amine LTs, feruloyl amide (FA) and coumaroyl amide (CA). These inhibitors are important because these two alone are sufficient to recapitulate the inhibitory effects of all LTs present. Analysis of the metabolome in untreated versus treated cells indicated that these phenolic amides cause rapid accumulation of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis. Moreover, isotopic tracer studies confirmed that carbon and nitrogen flux into nucleotides is inhibited by the amides, suggesting that these phenolic amines are potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Biochemical studies showed that the amides directly inhibit glutamine amidotransferases, with FA acting as a competitive inhibitor of the E. coli enzyme responsible for the first committed step in de novo purine biosynthesis. Supplementation of cultures with nucleosides was sufficient to reverse the effect of the amides, suggesting the ability to bypass the block in de novo nucleotide biosynthesis via salvage pathways. Collectively, these results provide a direct mechanism for the inhibitory effects of phenolic amides, knowledge that will inform future design of biocatalysts for improved bioconversion.

Reference: Pisithkul, T., T. B. Jacobson, T. J. O'Brien, D. M. Stevenson, and D. Amador-Noguez. 2015. “Phenolic Amides are Potent Inhibitors of De Novo Nucleotide Biosynthesis,” Applied and Environmental Microbiology 81(17), 5761-72. DOI: 10.1128/AEM.01324-15. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 14, 2015

N2O Emissions During Establishment Phase of Various Bioenergy Cropping Systems

As bioenergy cropping systems are developed, their greenhouse gas (GHG) emissions will be a key component of sustainability evaluations. Nitrous oxide (N2O) is a potent GHG and a substantial proportion of the total GHG footprint associated with feedstock production. N2O emitted from soils is primarily the result of microbial activities, which are influenced by various environmental factors including temperature and oxygen and water availability. The impact of each of these factors differs among various cropping systems. To understand how traditional and biomass feedstock cropping systems might vary with regard to N2O emissions, researchers at the Department of Energy’s Great Lakes Bioenergy Research Center compared the establishment phase N2O emissions of annual monocultures of continuous corn and corn-soybean-canola rotations; perennial monocultures of switchgrass, Miscanthus, and hybrid poplar; and perennial polycultures of early successional species, native grasses, and native prairie species. Measurements were done over a 2- to 4-year period following planting over which several perennial crops attained “full capacity” biomass production. They found that during the establishment phase, perennial bioenergy crops emit less N2O than annual crops, especially when not fertilized. Emissions for perennials were about three times less than for annuals on a per hectare basis. N2O peak fluxes were associated with periods of rain following fertilizer application. And finally, the results show that simulation models trained on single systems performed well in most monocultures but worse in polycultures, which means models including N2O emissions should be parameterized specifically for particular plant systems. The results suggest that perennial biomass feedstock cropping systems have the potential for a lower GHG burden even during their establishment phase.

Reference: Oates, L. G., D. S. Duncan, I. Gelfand, N. Millar, G. P. Robertson, and R. D. Jackson. 2015. “Nitrous Oxide Emissions During Establishment of Eight Alternative Cellulosic Bioenergy Cropping Systems in the North Central United States,” Global Change Biology Bioenergy, DOI: 10.1111/gcbb.12268. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 12, 2015

Using Natural Microbial Communities as Biosensors for Environmental Contaminants

Microbial communities are highly attuned to changes in environmental conditions, rapidly sensing and responding to shifts in temperature, pH, nutrient availability, toxin levels, and dozens of other variables. For decades, scientists have studied the abilities of microbes to survive exposure to (and in some cases make use of) environmental contaminants such as heavy metals, radionuclides, and hydrocarbons. However, microbial communities can contain hundreds of different species, and this complexity makes it extremely difficult to quantitatively measure community-level responses to contaminant exposure. In a new study, a team of researchers from Lawrence Berkeley National Laboratory’s ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) science focus area developed a new computational approach for the analysis and computational modeling of microbial community responses to environmental contaminants. Using direct sequencing of DNA from environmental samples, the team examined the microbial community of a subsurface aquifer in Oak Ridge, Tennessee, that had been contaminated with uranium, nitrate, and a variety of other compounds. Drawing on this data, a modeling framework was constructed to enable prediction of the types and amounts of contaminants that had been experienced by the microbial community based on known physiological characteristics of detected bacterial species. The predictions of this model strongly correlated with amounts of uranium, nitrate, and a variety of other geochemical factors measured at the sampling sites. To test the utility of this approach using an independent dataset, the team applied the model to microbial DNA samples collected during the Deepwater Horizon oil spill in 2010. Again, the model accurately predicted which samples had experienced oil contamination based on microbial DNA sequences and suggested that the community fingerprint retained a “memory” of exposure even after oil was no longer detectable. The results of this study provide a powerful new approach for not only the identification of contaminants in environmental samples, but also the microbial processes that are acting on them and potentially impacting their movement and/or longevity in the environment.

Reference: Smith, M. B., A. M. Rocha, C. S. Smillie, S. W. Oleson, C. J. Paradis, L. Wu, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, J. Phillips, S. M. Techtmann, D. C. Joyner, S. P. Preheim, M. S. Sanders, J. Yang, M. A. Mueller, S. C. Brooks, D. B. Watson, P. Zhang, Z. He, E. A. Dubinsky, P. D. Adams, A. P. Arkin, M. W. Fields, J. Zhou, E. J. Alm, and T. C. Hazen. 2015. “Natural Bacterial Communities as Quantitative Biosensors,” mBio 6(3), e00326-15. DOI: 10.1128/mBio.00326-15. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


May 01, 2015

Mechanisms of Limonene Toxicity and Tolerance Elucidated

Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. Limonene has antimicrobial properties, but also has potential as a biofuel component, making it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity was unknown. Researchers at the Department of Energy’s Joint BioEnergy Institute have characterized a limonene-tolerant strain of Escherichia coli and found a mutation in a gene encoding alkyl hydroperoxidase, which alleviates limonene toxicity. They found that the acute toxicity previously attributed to limonene was largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. The researchers found that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. These results have implications for both the renewable production of limonene and limonene’s applications as an antimicrobial.

Reference: Chubukov, V., F. Mingardon, W. Schackwitz, E. E. Baidoo, J. Alonso-Gutierrez, Q. Hu, T. S. Lee, J. D. Keasling, and A. Mukhopadhyay. 2015. “Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC,” Applied and Environmental Microbiology 81, 4690-6. DOI: 10.1128/AEM.01102-15. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 23, 2015

Using Metatranscriptomics to Understand Carbon Decomposition in Forest Soils

Decomposition of plant materials in soils is accomplished by a complex and highly diverse community of microorganisms. The vast majority of these microbes cannot be grown in laboratories, and the roles of different species in decomposition and responses to changing environmental conditions are not well understood. Ecologists have demonstrated that the addition of nitrogen to forest soils significantly slows the rate of carbon decomposition, but it is not well understood why this change occurs. Recent advances in soil metatranscriptomics, the direct analysis of microbial community gene expression in environmental samples, have provided researchers with a more sophisticated set of tools to track changes in microbial community structure and function. In a new study, a collaborative team of scientists at Los Alamos National Laboratory and the University of Michigan have completed a metatranscriptomic analysis of forest soils at a long-term ecological experiment examining impacts of nitrogen addition. By developing a new technique for metatranscriptomic sampling, the team was able to complete a much deeper analysis of community metabolic potential than has been previously attempted. Using this approach, fungal and bacterial genes involved in degradation of plant lignocellulose were determined to undergo large changes in expression at two separated sites with elevated nitrogen. Overall pattern shifts were consistent with decreased carbon decomposition rates, but specific mechanisms appeared to vary between the different forest sites. As climate change processes shift environmental variables and agricultural practices continue to alter nitrogen inputs in terrestrial soils, understanding their coupled impacts on microbial community activities will be crucial to more confidently modeling and predicting impacts on different ecosystems.

Reference: Hesse, C. N., R. C. Mueller, M. Vuyisich, L. Gallegos-Graves, C. D. Gleasner, D. R. Zak, and C. R. Kuske. 2015. “Forest Floor Community Metatranscriptomics Identify Fungal and Bacterial Response to N Deposition in Two Maple Forests,” Frontiers in Microbiology, DOI: 10.3389/fmicb.2015.00337. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 20, 2015

Hardwood Lignin Engineered into Softwoods

Conifer (softwood) biomass naturally has high lignin content and is more difficult to process than biomass from hardwood species because softwoods lack syringyl units in their lignins. Using genetic engineering strategies, researchers from the Department of Energy’s Great Lakes Bioenergy Research Center transformed into Pinus radiata two enzyme functions necessary to produce syringyl units in order to metabolicly engineer syringyl lignin production into conifers. Analytical methods performed on the transformed P. radiata showed evidence that the new enzymatic activities were being expressed—namely, ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT)—and that sinapyl alcohol was being incorporated into the lignin polymer. These results provide the proof of concept that generating a lignin polymer containing syringyl units is possible in softwood species such as P. radiata. Additionally, these results suggest that retaining the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods more favorable for industrial processing also may be possible.

Reference: Wagner, A., Y. Tobimatsu, L. Phillips, H. Flint, B. Geddes, F. Lu, and J. Ralph. 2015. “Syringyl Lignin Production in Conifers: Proof of Concept in a Pine Tracheary Element System,” Proceedings of the National Academy of Sciences (USA), DOI: 10.1073/pnas.1411926112. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 15, 2015

Determining Sugar Content of Plant Biomass

Assessing biomass recalcitrance in large populations of both natural and transgenic plants is important to identify promising candidates for lignocellulosic biofuel production. To properly test and optimize biofuel production parameters, the starting sugar content must be known to calculate percent sugar yield and conversion efficiencies. The current standard procedure is both labor- and time-intensive, requiring gram quantities of biomass and taking close to 2 weeks for the full analysis. Pyrolysis molecular beam mass spectrometry (py-MBMS) has been used as a high-throughput method for determining lignin content and structure, and researchers at the Department of Energy’s BioEnergy Science Center are demonstrating its applicability for deter­mining glucose, xylose, arabinose, galactose, and mannose content in biomass. Py-MBMS measure­ments of sugars in the biomass from conifers, hardwoods, and herbaceous species give similar values to those measured using standard high-performance liquid chromato­graphy, indicating that py-MBMS provides an accurate quantification of total sugar content for a range of biomass types. With data collection for py-MBMS taking only 1.5 minutes per sample, py-MBMS is a rapid high-throughput method for quantifying sugar content in biomass. This improved rate of analysis will help in evaluating approaches to overcoming biomass recalcitrance.

Reference: Sykes, R. W., E. L. Gjersing, C. L. Doeppke, and M. F. Davis. 2015. “High-Throughput Method for Determining the Sugar Content in Biomass with Pyrolysis Molecular Beam Mass Spectrometry,” BioEnergy Research, DOI: 10.1007/s12155-015-9610-5. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 15, 2015

Heterologous Orthogonal Fatty Acid Biosynthesis System in Escherichia coli for Oleochemical Production

Producing biofuels and bioproducts from biomass requires the construction of efficient biosynthetic pathways. The introduction of heterologous enzymes into the well-established model microbe, Escherichia coli, can have the benefits of expanding the metabolite produced while avoiding feedback inhibition. Researchers at the Department of Energy’s Joint BioEnergy Institute expressed several heterologous type I fatty acid synthases (FAS) in E. coli that functioned in parallel with the native FAS. The most active heterologous FAS expressed in E. coli was Corynebacterium glutamicum FAS1A and resulted in the production of oleochemicals including fatty alcohols and methyl ketones. Chain length distribution of fatty alcohols produced shifted with coexpression of FAS1A with the acyl carrier protein/coenzyme A (CoA)-reductase from Marinobacter aquaeolei (Maqu2220). Coexpression of FAS1A with the Micrococcus luteus acyl-CoA-oxidase (FadM, FadB) resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work is believed to be the first example of in vivo function of a heterologous FAS in E. coli. Functional expression of these large enzyme complexes in E. coli will enable their study without the need to culture the native organisms as well as enable the study of FAS from uncultured organisms. In addition, using FAS1 enzymes for oleochemical production has several potential advantages, and further optimization of this system could lead to strains with more efficient conversion of biomass to desired biofuels and bioproducts.

Reference: Haushalter, R. W., D. Groff, S. Deutsch , L. The, T. A. Chavkin, S. F. Brunner, L. Katz, and J. D. Keasling. 2015. “Development of an Orthogonal Fatty Acid Biosynthesis System in Escherichia coli for Oleochemical Production,” Metabolic Engineering 30, 1-6. DOI: 10.1016/j.ymben.2015.04.003. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 14, 2015

Methane Consumption by Microbes in High Arctic Soils

As global climate change warms Arctic ecosystems, organic carbon locked in frozen soils thaws and becomes susceptible to decomposition by microbes. Major uncertainties remain regarding what fraction of this carbon will be released as carbon dioxide (CO2) versus methane (CH4), especially in different types of environments. Both CO2 and CH4 act as greenhouse gases, but with different intensities and residence times in the atmosphere. Various microbes can either produce methane (methanogens) or consume it (methanotrophs), so understanding the roles played by these organisms in different Arctic habitats is critical in determining potential outcomes of warming scenarios. In a recent study, a collaborative team of researchers used a combination of systems biology tools and biogeochemical process measurements to examine methanogenic and methanotrophic microbes in soils on Axel Heiberg Island in the Canadian high Arctic. In a surprising finding, the low nutrient mineral soils found on the island acted a methane sink, actively removing CH4 from the atmosphere. Metagenomic profiling of core samples taken from these soils identified a specific subclass of high-affinity methanotrophs capable of growth on very low CH4 concentrations. Targeted metatranscriptomic and metaproteomic profiling demonstrated that these organisms are not only present in these samples, but are actively expressing the genes and protein involved in high-affinity CH4 uptake. In a series of microcosm experiments using intact soil cores from the island, the team subjected the samples to warming and moisture additions consistent with current climate change projections for the region. Although rates of CH4 production by methanogens increased in deeper layers of the samples, there was no net release of CH4, suggesting that it was completely consumed by methanotrophs and converted to CO2. These results are very different from observations in more nutrient-rich permafrost ecosystems, where warming typically results in significant CH4 releases. As predictions of climate change impacts continue to improve, these findings highlight the importance of understanding the complex set of interrelationships between microbial community members and habitat-specific environmental conditions.

Reference: Lau, M. C. Y., B. T. Stackhouse, A. C. Layton, A. Chauhan, T. A. Vishnivetskaya, K. Chourey, J. Ronholm, N. C. S. Mykytczuk, P. C. Bennett, G. Lamarche-Gagnon, N. Burton, W. H. Pollard, C. R. Omelon, D. M. Medvigy, R. L. Hettich, S. M. Pfiffner, L. G. Whyte, and T. C. Onstott. 2015. “An Active Atmospheric Methane Sink in High Arctic Mineral Cryosols,” The ISME Journal, DOI: 10.1038/ismej.2015.13. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 30, 2015

Promoter Set for Heterologous Gene Expression in Clostridium thermocellum

For successful fermentation of biofuels and bioproducts from biomass, using microorganisms for which fewer genetic tools have been developed might be the most effective approach. To date, most metabolic engineering work in Clostridium thermocellum has focused on gene deletion, but many metabolic engineering strategies require well controlled heterologous gene expression, which requires a collection of well characterized and understood promoters. Researchers from the Department of Energy’s BioEnergy Science Center sought to identify new promoters for predictable gene expression in C. thermocellum. For this work, 17 different C. thermocellum promoters were tested with two different reporter genes (LacZ and AdhB) to ensure the activity of the target promoter was not gene-specific. Putative promoters were chosen by analyses of published C. thermocellum gene expression datasets. Promoter activity in both C. thermocellum and Escherichia coli were testedbecauseideally a promoter would not be strongly expressed in E. coli to avoid toxicity problems during cloning. Several useful promoters were identified (eno, cbp, cbp_2, 815, 966, 2638, and 2926), which showed high expression and high enzymatic activity of both reporter genes in C. thermocellum. Other promoters were not useful, showing no heterologous gene activity or negatively impacting plasmid stability. These results provide several new good promoters for C. thermocellum. This improved understanding of promoter function will enhance efforts to express heterologous genes important for improved biofuel production in C. thermocellum.

Reference: Olson, D. G., M. Maloney, A. A. Lanahan, S. Hon, L. J. Hauser, and L. R. Lynd. 2015. “Identifying Promoters for Gene Expression in Clostridium thermocellum,” Metabolic Engineering Communications, DOI: 10.1016/j.meteno.2015.03.002. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 30, 2015

The Priming Effect: How Plant Root Exudates Make Soil Carbon More Susceptible to Microbial Degradation

Rates of decompositional processes performed by soil microbes are influenced by a variety of factors including temperature, water availability, and the presence of minerals. As plant materials are broken down by microbes, released organic carbon compounds can bind to soil minerals, becoming much less accessible to further decomposition. These bound pools of organic carbon can be stored in soils for years, decades, or centuries depending on local site conditions. However, microbiologists have long observed a phenomenon known as “the priming effect,” in which the addition of small amounts of unbound organic carbon results in microbial degradation of older pools of mineral-bound soil carbon. Elevated atmospheric CO2 levels recently have been shown to cause plant roots to increase their secretion of small carbon molecules (“exudates”), which has significantly increased the importance of understanding how the priming effect works. In a recent study, a team of scientists co-led by Lawrence Livermore National Laboratory and Oregon State University used a combination of microbial community analysis and high-resolution mass spectrometry (NanoSIMS) to examine the mechanistic basis of the priming effect in soil microcosms. When a variety of different carbon compounds associated with root exudates were added to the soils via an artificial root system, they were shown to directly disrupt associations between older carbon and soil minerals. Liberated carbon was rapidly consumed by soil microbes, and the team was able to follow correlated shifts in microbial community composition and elevated CO2 production. Different types of exudate compounds had varying degrees of ability to strip stored carbon from minerals, a particularly significant observation since elevated atmospheric CO2 shifts both the amounts and types of exudates that plants produce. These results represent a new breakthrough in understanding the molecular-scale mechanisms underlying the priming effect and could significantly advance our ability to predict impacts of climate change on carbon cycling in terrestrial ecosystems.

Reference: Keiluweit, M., J. J. Bougoure, P. S. Nico, J. Pett-Ridge, P. K. Weber, and M. Kleber. 2015. “Mineral Protection of Soil Carbon Counteracted by Root Exudates,” Nature Climate Change, DOI: 10.1038/NCLIMATE2580. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 27, 2015

Making Sense of Genomic Networks

Genomes contain the information underlying an organism’s molecular functions. One way to compare the entire genomes of different organisms is to compare their gene-family content profiles, which is effectively a comparison of their functional potential. Standard networks, when used to model phylogenomic similarities, are not capable of capturing some of the underlying complexity of the relationships between genomes. To address this limitation, scientists at Oak Ridge National Laboratory, funded through the Department of Energy’s Plant-Microbe Interfaces Science Focus Area, developed a new three-way similarity metric and constructed three-way networks modeling the relationships among 211 bacterial genomes. They found that such three-way networks find cross-species genomic similarities that would otherwise have been missed by simpler models such as standard networks. Interactions within and between the multiple species that make up the complex microbial communities associated with plant roots are believed to influence the plant’s overall health and vigor and may contribute to the plant’s ability to survive adverse environmental conditions. This research is the first time the concept of three-way networks has been applied in the field of comparative genomics. These networks will be a useful tool to model and reveal complex interspecific bacterial relationships that are not found using the conventional two-way network models, and could pave the way toward deciphering intricate plant-microbe and microbe-microbe interactions.

Reference: Weighill, D. A., and D. A. Jacobson. 2015. “Three-Way Networks: Application of Hypergraphs for Modelling Increased Complexity in Comparative Genomics,” PLoS Computational Biology 11(3), e1004079. DOI: 10.1371/journal.pcbi.1004079. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 23, 2015

New Technology Tracks Cells Containing Multiple Mutations Within a Cellular Population

Different techniques to generate large collections of cells intentionally mutated in a number of targeted genes are currently available, and specific mutants in those collections can be readily identified. However, to manipulate complex traits involving multiple genes, it is necessary to identify individual cells that contain several mutated genes. Tracking individual cells that harbor specific combinations of two or more mutations separated by long distances within their genome is a time-consuming and effort-intensive process. In a recent study, researchers at the University of Colorado in Boulder reported the development of a new method called "TRACE" that allows the identification of single bacterial or eukaryote cells with mutations in about six targeted genes. The technique uses mathematical modeling to design short DNA fragments (or primers) that specifically bind to the targeted mutation sites. These primers are synthesized in a way that allows amplification of the targeted regions and subsequent joining of the amplification products into a single DNA molecule. By performing the amplification and joining of the DNA products in an emulsion where each cell in the population is confined to a single droplet, the six targeted sites can be analyzed by high-throughput sequencing to identify which cells contain mutations in one or more of the sites. In proof-of-concept experiments, the team used TRACE to identify a combination of mutant genes that confer the bacterium Escherichia coli tolerance to the toxicity of cellulose hydrolysate and the biofuel isobutanol. Because of the much higher throughput of TRACE relative to other genotyping methods, this technology will substantially accelerate the engineering of microbes for the production of biofuels and other chemicals.

Reference: Zeitoun, R. I., A. D. Garst, G. D. Degen, G. Pines, T. J. Mansell, T. Y. Glebes, N. R. Boyle, and R. T. Gill. 2015. “Multiplexed Tracking of Combinatorial Genomic Mutations in Engineered Cell Populations,” Nature Biotechnology, DOI: 10.1038/nbt.3177. (Reference link)

Contact: Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 10, 2015

Comparative Genomics Reveal Functional Diversification of the Methanogen Methanosarcina mazei

Methanogenic archaea play a major role in global carbon cycle processes, participating in the conversion of organic carbon to the greenhouse gas methane in oxygen-limited environments such as waterlogged soils and wetland sediments. Different types of methanogens are capable of converting either hydrogen and carbon dioxide or intermediate fermentation products (e.g., ace­tate and methanol) into methane; both processes are key components of carbon decomposi­tion food webs. In a new study, researchers at the University of Illinois have completed a compara­tive genomics study on 56 different isolates of the metabolically versatile methanogen Methanosarcina mazei cultivated from sediments of the Columbia River in Oregon. While all isolates are members of the same species, they showed a surprising degree of genomic diversity and formed a distinct pattern of subgroups (i.e., clades) based on their site of isolation. The investigators were able to identify a core genome shared by all isolates, but other genetic elements were variable in distribution and showed evidence of transfer between different clades of M. mazei. Several of the variable genes encoding proteins involved the methanogenic metabol­ism, cofactor utilization, and (most intriguingly) uptake of organic substrates. These observations led the researchers to hypothesize that M. mazei has evolved into strains optimized for specific ecological niches in the sedimentary environments, a phenomenon that has been observed in environmental populations of bacteria. This hypothesis was supported by physiological experi­ments showing that isolates from different M. mazei clades varied in their ability to use the organic compound trimethylamine for methanogensis. These results advance our mechanistic understanding of a key step in the global carbon cycle and highlight the importance of analyzing metabolically significant differences that occur in microbes at the subspecies level.

Reference: Youngblut, N. D., J. S. Wirth, J. R. Henriksen, M. Smith, H. Simon, W. W. Metcalf, and R. J. Whitaker. 2015. “Genomic and Phenotypic Differentiation Among Methanosarcina mazei Populations from Columbia River Sediment,” The ISME Journal, DOI: 10.1038/ismej.2015.31. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 09, 2015

New Antifungal Agents from Lignocellulose Hydrolysate

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides to protect bioenergy crops. Scientists at the Department of Energy’s Great Lakes Bioenergy Research Center discovered that poacic acid found in lignocellulosic hydrolysates of grasses functions as a potent antifungal compound. Several lines of evidence pointed toward fungal cell wall synthesis as the point of action of poacic acid. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. In addition, morphological analysis of cells treated with poacic acid revealed morphologies similar to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development on soybean leaves. The discovery of poacic acid as a natural antifungal agent targeting ß-1,3-glucan further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates. This research highlights the potential use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds.

Reference: Piotrowski, J. S., H. Okada, F. Lu, S. C. Li, L. Hinchman, A. Ranjan, D. L. Smith, A. J. Higbee, A. Ulbrich, J. J. Coon, R. Deshpande, Y. V. Bukhman, S. McIlwain, I. M. Ong, C. L. Myers, C. Boone, R. Landick, J. Ralph, M. Kabbage, and Y. Ohya. 2015. “Plant-Derived Antifungal Agent Poacic Acid Targets ß-1,3-Glucan,” Proceedings of the National Academy of Sciences (USA), DOI: 10.1073/pnas.1410400112. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 02, 2015

Regulation of Lipid Accumulation in a Photosynthetic Bacterium

Lipids serve important functions in living systems, either as structural components of membranes or as a form of carbon storage. Understanding the mechanisms of lipid accumulation in microorganisms is important for providing insight into the assembly of biological membranes and additionally has important applications in the production of renewable fuels and chemicals. Researchers at the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) in collaboration with DOE’s Environmental Molecular Sciences Laboratory (EMSL) have investigated the ability of Rhodobacter sphaeroides to increase membrane production at low O2 tensions in order to house its photosynthetic apparatus. They found that this bacterium has a mechanism to increase lipid content in response to decreased O2 tension and identified a specific transcription factor necessary for this response. This finding is significant because it identifies a transcriptional regulatory pathway that can increase microbial lipid content and has applications for increasing biofuel production

Reference: Lemmer, K. C., A. C. Dohnalkova, D. R. Noguera, and T. J. Donohue. 2015. “Oxygen Dependent Regulation of Bacterial Lipid Production,” Journal of Bacteriology 197(9), 1649-58. DOI: 10.1128/JB.02510-14. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


March 02, 2015

Understanding and Enhancing Microbial Lipid Production for Biofuels

Lipids derived from oil-rich microorganisms such as bacteria, yeast, and microalgae offer a promising source of renewable fuels and chemicals. However, genetic and biochemical mechanisms regulating lipid accumulation in microorganisms are poorly understood. A recent study revealed a novel molecular pathway involved in microbial lipid accumulation. Researchers from the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) and the University of Wisconsin-Madison used the cryotransmission electron microscope at the DOE Environmental Molecular Sciences Laboratory to study lipid accumulation in the microbe Rhodobacter sphaeroides. Using fatty acid levels to assess membrane lipid content, the team found that the total fatty acid content per cell increased three-fold under low oxygen and anaerobic conditions compared to high oxygen conditions. They also found that the microbes’ lipid and pigment accumulation processes were separable, and they identified a transcription factor called PrrBA that is required for fatty acid accumulation in response to low oxygen levels. This new approach to maximize lipid production through an alteration in the activity of a single transcriptional regulator could lead to the development of strategies for engineering this microbe to increase yields for large-scale production of lipids for biofuels and chemicals.

Reference: Lemmer, K. C., A. C. Dohnalkova, D. R. Noguera, and T. J. Donohue. 2015. “Oxygen-Dependent Regulation of Bacterial Lipid Production,” Journal of Bacteriology, DOI: 10.1128/JB.02510-14. (Reference link)

Contact: Paul E. Bayer, SC-23.1, (301) 903-5324, Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 26, 2015

Novel Noncatalytic Cellulase-Binding Proteins Identified in Caldicellulosiruptor

Lignocellulose-degrading microorganisms often produce cellulosomes, which are protein complexes containing cellulase enzymes and noncatalytic binding modules. However, the genus Caldicellulosiruptor does not encode for cellulosomes, indicating that this genus uses alternative attachment mechanisms. To look for cellulose-binding proteins in Caldicellulosiruptor kronotskyensis, researchers from the Department of Energy’s BioEnergy Science Center performed a proteomic screen to detect proteins enriched in a cellulose-bound fraction. A comparison of amino acid sequences from the cellulose-binding proteins to the C. kronotskyensis genomic sequence identified the likely encoding gene and a closely related gene. These genes, subsequently named tapirins, are unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. In addition, no genes homologous to these tapirin genes were found outside of the genus Caldicellulosiruptor. Heterologously expressed tapirin gene products demonstrated binding to insoluble substrates such as Avicel, switchgrass, and Populus biomass, with a high affinity and specificity. Crystallization of a cellulose-binding truncation from one tapirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face and are structurally unique and define a new class of polysaccharide adhesins. Thus, the tapirins establish a new paradigm for how cellulolytic bacteria adhere to cellulose and may be used in engineering more efficient cellulase enzymes for more efficient lignocellulose deconstruction.

Reference: Blumer-Schuette, S. E., M. Alahuhta, J. M. Conway, L. L. Lee, J. V. Zurawski, R. J. Giannone, R. L. Hettich, V. V. Lunin, M. E. Himmel, and R. M. Kelly. 2015. “Discrete and Structurally Unique Proteins (Tapirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose,”The Journal of Biological Chemistry, DOI: 10.1074/jbc.M115.641480. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 23, 2015

Elucidating the Evolution of Mutualistic Plant Fungi

The large variety of fungi that exist in forest soils play diverse and important roles when in association with plant roots. One such type, the ectomycorrhizal (ECM) fungi, is a beneficial mutualist. ECM fungi obtain carbon compounds from the host plant, and in doing so provide critical ecological services such as decomposing lignocellulose and promoting plant growth. To unravel the mechanisms of nutrient cycling in forests, a better understanding of ECM fungi is needed. As part of a consortium investigating mycorrhizal fungal genomics, scientists at Oak Ridge National Laboratory, funded through the Department of Energy’s (DOE) Plant-Microbe Interfaces Science Focus Area, and DOE’s Joint Genome Institute performed phylogenomic and comparative genomic analyses of newly sequenced fungal genomes, including 13 ECM fungi, to elucidate the genetic bases of mycorrhizal lifestyle evolution. They found that although the ECM fungi have a reduced complement of genes encoding plant cell-wall degrading enzymes, those enzymes that were retained made up a distinct suite, indicating that they possess diverse capabilities to decompose lignocellulose. They also found that the symbiosis that develops between ECM fungi and the host plant and contributes to plant development and immunity requires lineage-specific fungal genes, including genes that code for mycorrhiza-induced small secreted proteins. The researchers conclude that convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a “symbiosis toolkit”, with reduced numbers of plant cell-wall degrading enzymes and lineage-specific suites of mycorrhiza-induced genes. Studies designed to predict the response of ECM and other mycorrhizal fungi to fluctuations in the environment will benefit from these genomic resources.

Reference: Kohler, A., A. Kuo, L. G. Nagy, E. Morin, K. W. Barry, F. Buscot, B. Canbäck, C. Choi, N. Cichocki, A. Clum, J. Colpaert, A. Copeland, M. D. Costa, J. Doré, D. Floudas, G. Gay, M. Girlanda, B. Henrissat, S. Herrmann, J. Hess, N. Högberg, T. Johansson, H.-R. Khouja, K. LaButti, U. Lahrmann, A. Levasseur, E. A. Lindquist, A. Lipzen, R. Marmeisse, E. Martino, C. Murat, C. Y. Ngan, U. Nehls, J. M. Plett, A. Pringle, R. A. Ohm, S. Perotto, M. Peter, R. Riley, F. Rineau, J. Ruytinx, A. Salamov, F. Shah, H. Sun, M. Tarkka, A. Tritt, C. Veneault-Fourrey, A. Zuccaro, Mycorrhizal Genomics Initiative Consortium, A. Tunlid, I. V. Grigoriev, D. S. Hibbett, and F. Martin. 2015. “Convergent Losses of Decay Mechanisms and Rapid Turnover of Symbiosis Genes in Mycorrhizal Mutualists,” Nature Genetics 47, 410-15, DOI:, DOI: 10.1038/ng.3223. (Reference link)

Contact: Cathy Ronning, SC-23.2, (301) 903-9549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 15, 2015

Newly Identified Archaea Involved in Anaerobic Carbon Cycling

Archaea, a domain of single-celled microorganisms, represent a significant fraction of Earth’s biodiversity, yet much less is known about Archaea than bacteria. One reason for this lack of knowledge is relatively poor genome sampling, which has limited accuracy for the Archaeal phylogenetic tree. To obtain a better understanding of the diversity and physiological functions of members of the Archaea domain, a team of scientists from the University of California, Berkeley, The Ohio State University, Columbia University, Lawrence Berkeley National Laboratory, the Department of Energy’s (DOE) Joint Genome Institute, Pacific Northwest National Laboratory, and DOE Environmental Molecular Sciences Laboratory used genome-resolved metagenomics analyses to investigate the diversity, genome sizes, metabolic capabilities, and potential environmental niches of Archaea from the Rifle, Colorado, uranium mill tailings site. The team used DOE JGI to sequence DNA from Rifle sediment and groundwater samples, and they not only identified new sequences for more than 150 Archaea but were able to reconstruct the complete genomes of two Archaea that were demonstrated to be representative of two different phyla. Transcriptomic studies conducted using EMSL capabilities on one of these microbes demonstrate that they have small genomes and limited metabolic capabilities; however, these metabolic capabilities are associated with carbon and hydrogen metabolism. These results suggest that these Archaea are either symbionts or parasites that depend on other organisms for some of their metabolic requirements. This research approximately doubled the known genomic diversity of Archaea, reconstructed the first complete genomes for Archaea using cultivation-independent methods, and enabled an extensive revision of the Archaeal tree of life. In addition, these findings can be incorporated into genome-resolved ecosystem models to more accurately reflect the role played by Archaea in the global carbon cycle.

References: Castelle, C. J., K. C. Wrighton, B. C. Thomas, L. A. Hug, C. T. Brown, M. J. Wilkins, K. R. Frischkorn, S. G. Tringe, A. Singh, L. M. Markillie, R. C. Taylor, K. H. Williams, and J. F. Banfield. “Genomic Expansion of Domain Archaea Highlights Roles for Organisms from New Phyla in Anaerobic Carbon Cycling,” Current Biology 25(6), 690-701. DOI: 10.1016/j.cub.2015.01.014. (Reference link)
(See also)

Contact: Paul E. Bayer, SC-23.1, (301) 903-5324, Dan Drell, SC-23.2, (301) 903-4742, David Lesmes, SC 23.1, (301) 903-2977, Pablo Rabinowicz, SC-23.2 (301) 903-0379
Topic Areas:

Division: SC-23.1 Climate and Environmental Sciences Division, BER,SC-23.2 Biological Systems Science Division, BER


February 11, 2015

Use of Co-Solvent Saves on Cost and Enzymes

Production of cost-effective biofuels from lignocellulosic biomass must overcome lignocellulose recalcitrance. Current processes to release sugars for viable biochemical conversion to biofuels requires energy-intensive pretreatment and large amounts of expensive enzymes. Researchers from the Department of Energy’s BioEnergy Science Center (BESC) have discovered that a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) reduces enzyme costs dramatically, resulting in high sugar yields from hemicellulose and cellulose. CELF employs tetrahydrofuran (THF), which is miscible with aqueous dilute acid, and gives up to 95% of the theoretical yield of glucose, xylose, and arabinose from corn stover even when coupled with enzymatic hydrolysis at only 2 mg enzyme/g glucan—an unusually low concentration of enzymes. The unusually high saccharification with such low enzyme loadings can be attributed to very high lignin removal, which was evidenced by compositional analysis, fractal kinetic modeling, and scanning electron microscopy imaging. Subsequently, nearly pure lignin product was precipitated giving a clean lignin stream for valorization. THF was efficiently recovered and recycled by evaporation of the volatile solvent. Simultaneous saccharification of CELF-pretreated solids with low enzyme loadings and fermentation by Saccharomyces cerevisiae produced twice as much ethanol as that from dilute acid-pretreated solids after being optimized for corn stover. Thus, CELF offers efficient lignocellulosic biomass pretreatment and saccharification with reduced costs relative to current processes.

Reference: Nguyen, T. Y., C. M. Cai, R. Kumar, and C. E. Wyman. 2015 “Co-Solvent Pretreatment Reduces Costly Enzyme Requirements for High Sugar and Ethanol Yields from Lignocellulosic Biomass,” ChemSusChem, DOI: 10.1002/cssc.201403045. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 30, 2015

Systems Biology of a Cyanobacterial Chassis for Photosynthetic Biosynthesis

Cyanobacteria, a broadly distributed class of photosynthetic bacteria, are attractive candidates for development as “chassis organisms” for production of biofuels and other products. In comparison to photosynthetic algae, cyanobacteria grow more quickly, are capable of growth in a broad range of conditions, and possess much simpler (and thus more easily engineered) genomes. However, developing systems-level understanding of integrated metabolic networks in cyanobacteria will be necessary before more sophisticated bioengineering approaches can be applied to further optimize performance or more easily introduce new biosynthetic modules. A new study by researchers at Washington University examines systems biology properties of the recently discovered cyanobacterial strain Synechococcus elongatus UTEX 2973, which grows at double the rates of other members of this species under high light intensities. Using a comparative genomics approach, the team was able to identify a surprisingly small set of genetic differences between UTEX 2973 and slower growing S. elongatus strains, amounting to 55 amino acid substitutions and a small missing region encoding six genes seen in the slower growing strains. Leveraging capabilities at the Department of Energy’s Environmental Molecular Sciences Laboratory, these findings were validated using global proteomics analysis, confirming predicted amino acid substitutions and showing that UTEX 2973 is missing five of the six predicted proteins. Although these proteins are currently of unknown function, UTEX 2973 fails to form cytoplasmic glycogen granules observed during growth of the other strains. This observation suggests that UTEX 2973 may not store photosynthetically fixed carbon, but instead immediately uses it as substrate fueling accelerated growth. UTEX 2973 can be genetically manipulated using tools developed for related cyanobacterial strains, and the team currently is developing a mutant library to explore the specific mechanistic basis of the UTEX 2973’s rapid growth phenotype. These findings expand our knowledge of cyanobacterial systems biology and present Synechococcus elongatus UTEX 2973 as a promising potential biotechnological chassis organism for the direct conversion of sunlight and CO2 into biofuels and other compounds.

Reference: Yu, J., M. Liberton, P. F. Cliften, R. D. Head, J. M. Jacobs, R. D. Smith, D. W. Koppenaal, J. J. Brand, and H. B. Pakrasi. 2015. “Synechococcus elongatus UTEX 2973, a Fast Growing Cyanobacterial Chassis for Biosynthesis Using Light and CO2,” Scientific Reports 5: 8132. DOI: 10.1038/srep08132. (Reference link)

Contact: Joseph Graber, SC-23.2, (301) 903-1239
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 22, 2015

Using a Designer Synthetic Media to Study Inhibitors Effect in Biomass Conversion

The biofuels industry has devoted significant efforts to converting lignocellulosic substrates into sugars that can be fermented into biofuels or other bioproducts. However, one of the major bottlenecks for cost-effective conversion in biorefineries has been the fermentation inhibition of yeast or bacteria by pretreatment degradation products. To engineer microbial strains for improved conversion, it is important to understand the inhibition mechanisms that affect the fermentative organisms in the presence of a lignocellulosic hydrolysate. One way in which these processes can be understood is by developing a synthetic hydrolysate media with a composition similar to the one that will be found after pretreating lignocellulosic biomass. Researchers at the Department of Energy’s Great Lakes Bioenergy Research Center characterized the plant-derived decomposition products present in ammonia fiber expansion (AFEX) pretreated corn stover hydrolsate (ACH), and a synthetic hydrolysate (SH) was formulated based on that ACH composition. The SH was used to evaluate the inhibitory effects of various families of decomposition products during fermentation using Saccharomyces cerevisiae strain 424A (LNH-ST). The SH did not entirely match the ACH performance; however, the major groups of inhibitory compounds were identified and used for further evaluation and comparison. Their characterization showed that the compounds present in ACH that were most inhibitory to fermentation were nitrogenous compounds, especially amides, though this result is associated with a concentration effect, given that nitrogenous compounds were the most abundant. Comparing inhibition due to amides in AFEX pretreatment versus inhibition due to carboxylic acids and other compounds formed in alternative pretreatment methods, they discovered that amides are significantly less inhibitory to both glucose and xylose fermentation. This means that ACH would be easily fermentable by yeast without any further detoxification. These studies help to map where to focus research efforts to overcome pretreatment byproduct inhibition of fermentation.

Reference: Tang, X., L. daCosta Sousa, M. Jin, S. P. S. Chundawat, C. K. Chambliss, M. W. Lau, Z. Xiao, B. E. Dale, and V. Balan. 2015. “Designer Synthetic Media for Studying Microbial-Catalyzed Biofuel Production,” Biotechnology for Biofuels 8:1. DOI: 10.1186/s13068-014-0179-6. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 13, 2015

Diversion of Lignin Precursor Reduces Content and Improves Biomass Saccharification Efficiency

Lignin confers recalcitrance to plant biomass used for producing biofuels and bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically have employed gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, researchers at the Department of Energy’s Joint BioEnergy Institute (JBEI) employed a new strategy using gain of function. In this method, expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) was targeted to the plastids of Arabidopsis to convert 3-dehydroshikimate—an intermediate of the shikimate pathway—into protocatechuate. This enzymatic conversion diverted lignin precursor into protocatechuate and related molecules and away from lignin precursors. Compared to wild-type plants, Arabidopsis lines expressing QsuB contained reduced levels of lignin deposition in the cell walls. Because this strategy is a gain of function, its expression can be controlled by selective promoters, thus offering better spatiotemporal control of lignin deposition than the gene knockout or gene silencing strategies. Finally, biomass from these engineered Arabidopsis lines exhibits more than a twofold improvement in saccharification efficiency. This result confirms that QsuB expression in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.

Reference: Eudes, A., N. Sathitsuksanoh, E. E. Baidoo, A. George, Y. Liang, F. Yang, S. Singh, J. D. Keasling, B. A. Simmons, and D. Loqué. 2015. “Expression of a Bacterial 3-Dehydroshikimate Dehydratase Reduces Lignin Content and Improves Biomass Saccharification Efficiency,” Plant Biotechnology Journal, DOI: 10.1111/pbi.12310. (Reference link)

Contact: Kent Peters, SC-23.2, (301) 903-5549
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Division: SC-23.2 Biological Systems Science Division, BER