BER Research Highlights

Search Date: October 19, 2017

34 Records match the search term(s):


December 27, 2016

Microbial Community Interactions Drive Methane Consumption in Lakes

Understanding interactions among organisms in complex microbial communities sheds new light on globally significant environmental processes.

The Science  
Large amounts of methane, a potent greenhouse gas, are produced as a byproduct during decomposition of plant matter in the sediments of lakes and wetlands. Bacteria known as methanotrophs consume much of this methane before it can enter atmosphere. In a recent study, researchers examined community interactions among methanotrophs and other types of microbes that control this important process.

The Impact
The biological mechanisms underlying many important environmental processes can be understood only by examining cooperative processes performed by diverse communities of microbes. This study uses an elegantly constructed model experiment and genomic analysis to examine the genetic basis of these interactions and determine how they influence microbial consumption of methane in lake sediments.

Summary
Several decades of research have demonstrated the importance of bacterial methanotrophs in carbon cycling processes of lakes, wetlands, and a variety of other environments. However, methanotrophs exist as members of diverse communities of regularly co-occurring non-methanotrophic microbes, and the roles of these organisms in methane cycling are not well understood. In a recent study, researchers at the University of Washington assembled an experimental model community of methanotrophs and associated non-methanotrophic microbes previously isolated from lake sediments. Using a community-scale metaomics analysis of shifts in gene expression, the team tracked how the associated organisms influenced each other during methane-driven growth. The presence of non-methanotrophs was shown to trigger an enzymatic and metabolic shift in the methanotrophs, resulting in conversion of a portion of the available methane into methanol, which was released to fuel the growth of these microbes. Not yet clear is if the methanotrophs derive some form of reciprocal benefit from this “cross-feeding,” or if this represents a type of parasitism. In either case, these findings considerably alter current understanding of methanotrophy as it occurs in complex environmental communities and suggest that much remains to be learned about the basic biological mechanisms driving an important element of the global carbon cycle.

Contacts (BER PM)
Dr. Joseph Graber
DOE Office of Biological and Environmental Research, Biological Systems Science Division
joseph.graber@science.doe.gov

(PI Contact)
Dr. Mary Lidstrom
University of Washington
lidstrom@u.washington.edu

Funding
This study was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science program under award DE-SC-0010556.

Publication
S. M. B. Krause, T. Johnson, Y. S. Karunaratne, Y. Fu, D. A. C. Beck, L. Chistoserdova, and M. E. Lidstrom, “Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions.” Proceedings of the National Academy of Sciences (USA) 114(2), 358-63 (2017). DOI: 10.1073/pnas.1619871114. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Diverse microbial communities consume methane produced as a byproduct during decomposition of plant matter in lake sediments. Image courtesy of iStock



December 22, 2016

Metagenomics Leads to New CRISPR-Cas Systems

Researchers discover the first CRISPR-Cas9 system in archaea.

The Science
Using large amounts of metagenomic data generated by the Department of Energy’s Joint Genome Institute (DOE JGI), researchers analyzed 155 million protein coding genes from uncultivated microbial communities. This work led to the discovery of the first CRISPR- (clustered regularly interspaced short palindromic repeats) Cas9 protein in the archaeal domain, as well as two previously unknown simple bacterial CRISPR-Cas systems.

The Impact
Microbes play key roles in the planet’s cycles, and characterizing them helps researchers work toward solutions for energy and environmental challenges. Examining environmental microbial communities has enabled access to an unprecedented diversity of genomes and CRISPR-Cas systems that have many applications, including biological research. The combined computational-experimental approach that was successful in this study can be used to investigate nearly all environments where life exists.

Summary
Microbes heavily influence the planet’s cycles, but only a fraction have been identified. Characterizing the abundant but largely unknown extent of microbial diversity can help researchers develop solutions to energy and environmental challenges. In microbes, CRISPR-Cas systems provide a form of adaptive immunity, and these gene-editing tools are the foundation of versatile technologies revolutionizing research. Thus far, CRISPR-Cas technology has been based only on systems from isolated bacteria. In a study led by longtime DOE JGI collaborator Jill Banfield of the University of California, Berkeley, researchers discovered, for the first time, a CRISPR-Cas9 system in archaea, as well as simple CRISPR-Cas systems in uncultivable bacteria. To identify these new CRISPR-Cas systems, the team harnessed more than a decade’s worth of metagenomic data from samples sequenced and analyzed by DOE JGI, a DOE Office of Science user facility. The CasX and CasY proteins were found in bacteria from groundwater and sediment samples. The archaeal Cas9 was identified in samples taken from the Iron Mountain Superfund site as part of Banfield’s pioneering metagenomics work with DOE JGI. Both CasX and CasY are among some of the most compact systems ever identified. This application of metagenomics validates studies of CRISPR-Cas proteins using living organisms.

Contacts
Daniel Drell, Ph.D.
Program Manager
Biological Systems Science Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
daniel.drell@science.doe.gov

David Lesmes, Ph.D.
Program Manager
Climate and Environmental Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
david.lesmes@science.doe.gov

Jill Banfield
University of California, Berkeley
jbanfield@berkeley.edu

Funding
DOE Office of Science, National Science Foundation, EMBO, German Science Foundation, Paul Allen Institute, and Howard Hughes Medical Institute 

Publication
Burstein, D., et al., “New CRISPR-Cas systems from uncultivated microbes.” Nature (2016). [DOI: 10.1038/nature21059] (Reference link)

Related Links

Topic Areas:

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



An artistic representation of the Tree of Life, with the many groups of bacteria and archaea at the upper left and eukaryotes, which include humans, at the lower right. Department of Energy Joint Genome Institute scientists are using gene-editing tools to explore microbial “dark matter.” [Artistic representation of Fig. 1 from Hug et al., “A new view of the tree of life.” Nature Microbiology 1 (2016). DOI: 10.1038/nmicrobiol.2016.48. Courtesy of Jill Banfield, University of California, Berkeley]



December 12, 2016

A New High-Throughput Genome Editing Technique to Generate Mutant Bacterial Strains

Using computer-aided design to develop a CRISPR/Cas9-based approach to cause thousands of mutations and map their effects to the mutated genes.

The Science
The generation of large collections of mutant bacterial strains is limited due to low mutagenic efficiencies and the difficulty of tracking diverse types of mutations or their combinations. Researchers at the University of Colorado in Boulder and their collaborators have taken advantage of the high editing efficiency of the CRISPR (clustered regularly interspaced short palindromic repeats) -Cas9 system, combined with synthetic bar-codes, to develop a method that can mutate thousands of genes and easily track the mutated genes to determine their effect on the bacterial physiology.    

The Impact
This new editing technique, for the first time, makes it possible to induce individual mutations throughout a bacterial genome in parallel, and associate each mutation with the resulting phenotype at single-nucleotide resolution in a single experiment. This method gives researchers the ability to design and modify microorganisms in a genome-wide manner allowing them to engineer new metabolic pathways for the production of biofuels and other relevant industrial products.

Summary
A CRISPR-enabled trackable genome engineering (CREATE) cassette was developed to include a targeting guide RNA (gRNA), a DNA sequence homologous to a given target locus in the genome, and a unique bar code to tack each mutation. A computationally designed library of over 50,000 CREATE cassettes targeting multiple genome locations was synthesized and used to induce specific mutations in a bacterial population. The resulting mutant strains were tracked by genomic sequencing showing an average editing efficiency of 70%. The CREATE library was tested on a bacterial culture under thermal stress and several hundred mutants that had previously been identified as adaptations to heat were also identified with CREATE, in addition to 140 new mutations in genes involved in the bacterial response to high temperature. Furthermore, several strains that showed high stress tolerance were the result of combinations of two or more single-nucleotide mutations that would not have been detected in normal mutagenesis experiments. The potential of CREATE to identify improved mutant strains can be used to develop new and enhanced biosynthetic abilities for the biological production of fuels and relevant chemicals.

Contacts (BER PM)
Pablo Rabinowicz
Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
Ryan Gill
Department of Chemical and Biological Engineering
University of Colorado
Boulder, Colorado
rtggtr@me.com

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science award DE-SC0008812. The authors also acknowledge support from the CAPES foundation.

Publications
Andrew Garst, Marcelo Bassalo, Gur Pines, Sean Lynch, Andrea Halweg-Edwards, Rongming Liu, Liya Liang, Zhiwen Wang, Ramsey Zeitoun, William Alexander, and Ryan Gill, “Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.” Nature Biotechnology 35, 48 (2017). [DOI: 10.1038/nbt.3718] (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


December 06, 2016

New Software Tools Streamline DNA Sequence Design-and-Build Process

These enhanced tools will accelerate gene discovery and characterization.

The Science                       
Synthetic DNA enables scientists to expand the breadth and depth of their genomic research. In a recent study, researchers developed a suite of build optimization software tools (BOOST) to streamline the design-build transition in synthetic biology engineering workflows. BOOST can automatically detect “difficult” sequences of nucleotides and redesign them for DNA synthesis, addressing DNA sequences with certain problematic characteristics (e.g., extreme % guanine-cytosine content, sequence patterns, and repeats), which decrease the success rate of DNA synthesis.

The Impact
By improving the design and manufacture of synthetic DNA through enhanced tools, scientists can accelerate gene discovery and gene characterization toward practical applications for energy and the environment.

Summary
The ability to design and manufacture synthetic DNA has opened tremendous possibilities in genomic research. In addition to providing access to samples that are difficult to find in nature (as well as crafting genomic sequences not known to occur in the natural world), manufacturing DNA enables scientists to test any sequence in a wide variety of contexts and environments. Biological computer-aided design and manufacture (bioCAD/CAM) software tools help researchers design sequences that can be critical to discovering new solutions for energy and the environment. So far, however, the software has not been able to automatically fix problematic sequences, slowing down the transition from the design to the manufacturing process and delaying the synthesis of designed DNA.

To solve this problem, researchers at the U.S. Department of Energy’s (DOE) Joint Genome Institute (JGI), a DOE Office of Science user facility, developed the BOOST suite to automate the synthetic DNA design process—and do away with the trial-and-error process scientists currently utilize to determine a sequence that can be synthesized.

The new suite of tools is available as a web application, an executable JAVA Archive (JAR), and as a representational state transfer application program interface (RESTful API). Ultimately, BOOST will accelerate the use of synthetic DNAs to explore gene functions relevant to DOE’s energy and environmental missions.

Contact (BER PM)
Daniel Drell, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
US Department of Energy
daniel.drell@science.doe.gov

(PI Contact)
Samuel Deutsch
DOE Joint Genome Institute SDeutsch@lbl.gov

Funding
U.S. Department of Energy Office of Science

Publication
E. Oberortner, J.F. Cheng, N.J. Hillson, and S. Deutsch, “Streamlining the design-to-build transition with build-optimization software tools.” ACS Synthetic Biology (2016). DOI:10.1021/acssynbio.6b00200. (Reference link)

Related Links
BOOST
JGI: DNA Synthesis Science Program

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The goal of build optimization software tools is to streamline, in a scalable fashion, the process of designing readily synthesizable DNA fragments. [Image courtesy of the Department of Energy’s Joint Genome Institute]



November 29, 2016

A synthetic microbial ecosystem helps understand the behavior of bacterial communities


A bacterial co-culture was engineered to force two bacterial species to depend on each other to grow, shedding light on mutualism dynamics.

The Science
Researchers designed a stable co-culture in which Escherichia coli consumed sugar and produced organic acids to feed a Rhodopseudomonas palustris mutant strain that fixed and provided nitrogen for both microbes. A mathematical model was developed for this system, and the model accurately predicted how the co-culture would reach a new equilibrium when one of the microbes was genetically modified.

The Impact
Artificial co-cultures of two or more microbial species are useful tools for understanding how microbial communities behave in their natural habitat. However, the instability of co-culture systems has limited their utility for both fundamental and biotechnological studies. This research developed a microbial cross-feeding system that maintains its species composition over multiple generations, constituting a novel and important tool for understanding mutualistic relationships in natural environments and how to manipulate microbial communities for useful purposes.     

Summary
A mutant strain of R. palustris that can fix nitrogen gas and excrete ammonium was cultured together with E. coli in the presence of glucose as the only carbon source. R. palustris cannot consume glucose, but it feeds on the organic acids excreted by E. coli after it metabolizes glucose. In turn, E. coli obtains its nitrogen from the ammonium excreted by the R. palustris mutant. This cross-feeding dependency forced the co-culture to stabilize at a ratio of one E. coli cell to nine R. palustris cells, regardless of the proportion of each strain in the initial inoculum. The researchers at Indiana University also developed a mathematical model that enabled them to successfully predict the co-culture composition if the amounts of nutrients excreted by the microbes were altered. To test the model's accuracy, the investigators made a new R. palustris mutant that excreted three times more ammonium than the original strain. When this new mutant was co-cultured with E. coli, the system reached equilibrium at a ratio of one-to-one, as the model predicted. These results demonstrate the utility of stable co-cultures to understand cross-feeding relationships in ecosystems relevant for the global carbon cycle, or to engineer microbial systems for practical applications.

Contact (BER PM)
Pablo Rabinowicz
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
pablo.rabinowicz@science.doe.gov

(PI Contact)
James McKinlay
Joint BioEnergy Institute
Department of Biology
Indiana University
Bloomington, IN
jmckinla@indiana.edu

Funding
This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research Early Career Research Program award number DE-SC0008131 to James McKinlay. Authors also acknowledge partial support from the U.S. Army Research Office.

Publication
LaSarre, B., A. McCully, J. Lennon, and J. McKinlay. 2017. “Microbial Mutualism Dynamics Governed by Dose-Dependent Toxicity of Cross-Fed Nutrients,” The ISME Journal 11, 337–48. DOI: 10.1038/ismej.2016.141.

Reference link: http://www.nature.com/ismej/journal/v11/n2/abs/ismej2016141a.html.

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Microbial interactions, including mutualistic nutrient exchange, underpin the flow of energy and materials in all ecosystems. [Image courtesy J. B. McKinlay]



November 21, 2016

A Big Step Forward in Designing Drought-Tolerant Bioenergy Crops

Day and night patterns of gene activity in agave reveal key genes involved in a type of photosynthesis that maximizes water-use efficiency.

The Science
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis found in plants adapted to hot and arid conditions. CAM photosynthesis differs from the more common C3 and C4 photosynthesis types in that it inverts the day and night pattern of stomata opening to capture carbon dioxide (CO2) at night and avoid water evaporation through stomata opening during the day. Researchers at the University of Nevada and Oak Ridge National Laboratory conducted metabolomics, proteomics, and transcriptomics analyses of the desert plant agave across a diel cycle to identify genes involved in the CAM photosynthesis process and its higher water-use efficiency.

The Impact
As the photosynthetic machinery of most bioenergy crops is adapted to temperate and humid environments, carbon fixation and, therefore, biomass accumulation are less efficient and require more water than CAM plants adapted to hot and dry conditions. For that reason, introducing CAM photosynthesis into bioenergy crops would enable them to grow in marginal environments, but the molecular and genetic basis of CAM photosynthesis are not well enough understood to do this. This research identified candidate genes responsible for several aspects of the CAM process that can be used to design bioenergy crops with increased water-use efficiency and tolerance to extreme environmental conditions. 

Summary
A comparison of diel metabolic profiles of the CAM photosynthesis plant agave and the C3 photosynthesis plant Arabidopsis showed that metabolites involved in the redox reactions required for photosynthesis are found at different times of the day in each plant. Consistent with those results, transcription and protein profiling confirmed that the expression patterns of genes necessary for redox balance were shifted between agave and Arabidopsis through the day and night cycle. Furthermore, cell signaling genes in the guard cells that form the stomata, as well as CO2-sensing genes responsible for the closing of stomata and ion channels that participate in stomata opening, also showed the same opposite expression patterns between the two photosynthetic modes. This research provides strong evidence that bioengineering CAM in a C3 plant will require temporal reprogramming and identifies potential key targets for engineering this mode of photosynthesis in C3 plants, such as poplar and other selected bioenergy crops.     

Contacts (BER PM)
Pablo Rabinowicz
DOE Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contacts)
Xiaohan Yang
Biosciences Division
Oak Ridge National Laboratory, Oak Ridge, TN
yangx@ornl.gov

John Cushman
Department of Biochemistry and Molecular Biology
University of Nevada, Reno, NV
jcushman@unr.edu

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science award DE-SC0008834.

Publication
Abraham, P. E., H. Yin, A. M. Borland, D. Weighill, S. D. Lim, H. C. De Paoli, N. Engle, P.C. Jones, R. Agh, D. J. Weston, S. D. Wullschleger, T. Tschaplinski, D. Jacobson, J. C. Cushman, R. L. Hettich, G. A. Tuskan, and X. Yang. 2016. “Transcript, Protein, and Metabolite Temporal Dynamics in the CAM Plant Agave,” Nature Plants 2(16178), DOI: 10.1038/nplants.2016.178. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


November 16, 2016

Bacteria Living Within Plant Roots Affect Where and How Plants Allocate Carbon for Growth

Bacteria within plant root tissues influence the size and shape of plant leaves and roots, as well as how plants allocate carbon toward leaves, stems, or roots.

The Science
Plant traits, such as root and leaf area, influence how plants interact with their environment, and bacteria living within plant tissues can determine morphology (plant form and structure) and physiology (how they function). To understand how different microbes shaped plant morphology and physiology, researchers inoculated cottonwood seedlings with three different strains of root-dwelling bacteria. They found that the bacteria did not change photosynthesis rates or total biomass, but bacteria regulated where carbon was allocated and how plants used it. Additionally, the researchers found closely related bacteria can have vastly different effects on plant growth.

The Impact
Since plants interact with their environment through their traits, bacteria may be an important middleman in determining how plants will respond to changing environmental conditions.

Summary
Bacteria living within plant tissues (endophytes) can change how plants express traits such as root and leaf growth rates and the ratio of root to leaves. Small changes in these traits could build up to alter how plants survive, adapt, and compete within their environment. In a recent study, researchers either inoculated cottonwood seedlings with one of three endophytic bacterial stains or left the plant un-inoculated as a control. They then looked at several responses including root and leaf growth rate, plant biomass, photosynthetic rate, and the ratio of roots to leaves. They found that inoculation was linked to an increase in root and leaf growth rate, but that this increase in growth rate did not lead to an increase in plant biomass or photosynthetic efficiency. These findings indicate bacterial endophytes can change where and how carbon is used in a plant, but may not increase the overall amount of carbon fixed by photosynthesis and stored in the plant’s biomass.

Contacts (BER PM)
Daniel Stover, SC-23.1, Daniel.Stover@science.doe.gov, 301-903-0289

(PI Contact)
Aimee T. Classen      
University of Vermont
Aimee.Classen@uvm.edu

Funding
Funding was provided by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER), Genomic Science program as part of the Plant-Microbe Interfaces Scientific Focus Area project at Oak Ridge National Laboratory. Additional funding was provided by BER’s Terrestrial Ecosystem Science program under award number DE-SC0010562.

Publication
Henning, J., et al. 2016. “Root Bacterial Endophytes Alter Plant Phenotype, but not Physiology,” PeerJ  4, e2606. DOI: 10.7717/peerj.2606. (Reference link)

Topic Areas:

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


November 11, 2016

Nitrogen Uptake Between Fungi and Orchids

Fungal and plant gene expression provides clues to nitrogen pathways.

The Science                       
Orchids are an example of an experimentally tractable plant that is highly dependent on its relationship with its mycorrhizal fungal partners for nutrient supply. In a recent study, researchers, for the first time, identified some genetic determinants potentially involved in nitrogen uptake and transfer in orchid mycorrhizas.

The Impact
This study provides a model system, amenable to experimental manipulation, for plant-fungi nutrient exchanges on a symbiotic level. It also offers insights into how host plants benefit from the mutualistic relationships formed with soil fungi that can expand their habitat range. Understanding these vital relationships may shed light on microbial symbioses applicable to growing bioenergy feedstock plants.

Summary
Orchids, like the majority of terrestrial plants, form symbiotic relationships between their plant roots and soil fungi, known as mycorrhizal associations. However, unlike other terrestrial plants, orchids rely on their mycorrhizal fungal partners for nutrient supply during the feed germination and development stages. Following these stages, most orchid species develop leaves and are capable of self-nourishment, whereas some species continue to rely on their fungal partners for an organic carbon supply. In this study, a team led by University of Turin researchers investigated the orchid mycorrhizal fungus Tulasnella calospora as both a free-living mycelium and in symbiosis with the photosynthetic orchid long-lipped serapias, or Serapias vomeracea. For the first time, researchers looked at the fungal genes that may have been involved in both the uptake and transfer of nitrogen to the host plant. RNA sequencing for the project was performed at the U.S. Department of Energy’s (DOE) Joint Genome Institute (JGI), a DOE Office of Science user facility.

The team also used JGI’s fungal genome database MycoCosm to identify fungal genes coding for proteins that were involved in nitrogen uptake and transfer. They found that the T. calospora genome has two genes coding for ammonium transporters and several genes coding for amino acid transporters, proteins that play roles in the nitrogen nutrient pathway. Overall, the orchid mycorrhizal fungi’s use of nitrogen may broaden the habitat ranges of orchids, allowing them to grow in a variety of soil types. Of more general interest to the DOE, this study provides important insights for this process and furthers understanding of plant-microbial symbioses that are vital for plant health and may inform understanding of microbial symbioses relevant to bioenergy feedstock plants.

Contacts
Daniel Drell, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
daniel.drell@science.doe.gov

Silvia Perotto
University of Turin
silvia.perotto@unito.it

Funding
U.S. Department of Energy Office of Science
Ministry of Education, Universities, and Research (Italy)
University of Turin
‘Compagnia di San Paolo’ (Torino, Italy)
Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE)

Publication
Fochi, V., W. Chitarra, A. Kohler, S. Voyron, V. Singan, E. Lindquist, K. Berry, M. Girlanda, I. V. Grigoriev, F. Martin, R. Balestrini, and S. Perotto. 2017. “Fungal and Plant Gene Expression in the Tulasnella calosporaSerapias vomeracea Symbiosis Provides Clues About Nitrogen Pathways in Orchid Mycorrhizas,” New Phytologist 213(1), 365-79. DOI: 10.1111/nph.14279. (Reference link)

Related Links
JGI MycoCosm Fungal Genomic Resource
JGI MycoCosm Fungal Genomic Resource: Tulasnella calospora
JGI Community Science Plans FY 2013: The Mycorrhizal Genomics Initiative
JGI Community Science Plan FY 2016: Microbial Mutualism with Orchids
JGI MycoCosm Fungal Genomic Resource: Sebacina vermifera
JGI MycoCosm Fungal Genomic Resource: Sebacina vermifera
JGI MycoCosm Fungal Genomic Resource: Ceratobasidium

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Researchers investigated Tulasnella calospora as both a free-living mycelium and in symbiosis with Serapias vomeracea (pictured). [Image courtesy Ziegler175 Wikimedia Commons, CC BY-SA 3.0]



November 06, 2016

Consequences of Drought Stress on Biofuel Production

Switchgrass cultivated during a year of severe drought inhibited microbial fermentation.

The Science
Investment in plant-derived sustainable biofuel sources could contribute to a near-term solution toward U.S. energy security and independence. However, weather conditions have the potential to greatly affect yearly biomass production. When plants are grown under water-stressed conditions, reduction in photosynthesis and slower growth are exhibited, leading to decreased biomass production. In this study researchers examined the effect of weather on biofuel production by comparing switchgrass and corn stover harvested after a year of major drought and after 2 years of normal precipitation (2010 and 2013).

The Impact
The study is the first linking variation in environmental conditions during bioenergy crop growth to potential detrimental effects on fermentation during biofuel production. This underscores the need for the development of biofuel production systems able to tolerate changes in precipitation and water availability as well as robust fermentation processes.

Summary
In response to the 2012 severe Midwestern drought, soluble sugar accumulated in switchgrass at significantly higher levels in comparison to non-drought period years. These sugars were chemically changed during the pretreatment stage, the step which opens up the physical structure of the plant cell wall. The soluble sugars chemically changed by reacting with the ammonia-based pretreatment chemicals to form highly toxic compounds known as imidazoles and pyrazines. The formation of toxic compounds during the pretreatment stage inhibited conversion, the final step where intermediates such as sugars are fermented into biofuel by microorganisms, such as the microbe S. cerevisiae. However, it may be possible to overcome this issue by 1) removing the soluble sugars prior to pretreatment or 2) using microbial strains resistant to the toxic effects of imidazoles and pyrazines. This study demonstrates that while there are benefits to growing bioenergy crops on marginal lands to avoid competition with food crops, the plants grown there may experience higher levels of stress resulting in deleterious impacts on microbes during biofuel production. To develop sustainable biofuel production systems, the deleterious effects of stress, such as fluctuations in precipitation and water availability, must be mitigated. This research helps to provide an understanding of the effects of drought stress on switchgrass and is relevant to DOE’s energy and environmental missions.

Contact
Kent Peters, Ph.D.
Program Manager Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
U.S. Department of Energy
kent.peters@science.energy.doe.gov

(PI Contact)
Rebecca Garlock Ong
Assistant Professor, Chemical Engineering - Michigan Technological University
rgong1@mtu.edu

Funding
This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494). Additional funding for L.G.O. is under DOE OBP Office of Energy Efficiency and Renewable Energy (DE-AC05-76RL01830).

Publications
R.G. Ong, A. Higbee, S. Bottoms, Q. Dickinson, D. Xie, S.A.Smith, J. Serate, E. Pohlmann, A.D. Jones, J.J. Coon, T.K. Sato, G.R. Sanford, D. Eilert, L.G. Oates, J.S. Piotrowski, D.M. Bates, D. Cavalier, and Y. Zhang, “Inhibition of microbial biofuel production in drought-stressed switchgrass hydrolysate.” Biotechnology for Biofuels 9, 237 (2016) [DOI: 10.1186/s13068-016-0657-01] (Reference link)

Related Links
Great Lakes Bioenergy Research Center

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



From field to fuel: Illustration of switchgrass conversion process. [Image courtesy of Matthew Wishiewski]



October 26, 2016

New System for Introducing Genetic Pathways into Plants, Making Them More Productive

A yeast DNA recombination system facilitates the transference and expression of entire heterologous metabolic pathways into plant genomes.

The Science
Researchers have adapted a DNA recombination system from yeasts to facilitate the construction of large stretches of DNA and their introduction into plant genomes. This technology, called jStack, advances the engineering of new, complex functionality into plants by enabling the expression of heterologous multigene pathways.

The Impact
Engineering more productive and resilient crops requires the introduction of new biological functions into plants. Often, multiple genes from different organisms need to be transferred to a given crop to provide new desirable properties, but assembling and introducing multiple genes into plant crops is difficult. The jStack technology will make it easier to combine genes from different sources and incorporate them into the genome of engineered crops to improve their performance. 

Summary
Researchers at Lawrence Berkeley National Laboratory modified plasmid vectors commonly used for plant transformation so that they can be replicated and selected in yeasts, in addition to the Escherichia coli and plant hosts, to create a multigene plant transformation system called jStack. The system also includes yeast DNA sequences required for homologous recombination so that multiple DNA elements can be assembled into a single DNA molecule in yeast intermediary hosts in vivo. The resulting recombinant vectors can then be selected and purified for introduction in the desired plant host. In an attempt to standardize plant genetic engineering, the jStack system was designed to be compatible with commonly used cloning systems. Furthermore, a publicly available library of over 100 compatible promoters, genes, and terminator sequences was created to encourage collaboration and innovation within the plant synthetic biology community. The utility of the jStack technology was validated by introducing the entire pathway of the pigment violacein from the soil bacterium Chromobacterium violaceum into a model plant, as well as the metabolic pathway required to produce bisabolene, a precursor to bisabolane and a potential biodiesel component.    

Contacts (BER PM)
Pablo Rabinowicz
DOE Office Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
Dominique Loqué
Joint BioEnergy Institute
Biological Systems and Engineering Division
Lawrence Berkeley National Laboratory
Berkeley, CA
dloque@lbl.gov

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science Early Career Research Program award to D. Loqué, and by contract DE-AC02-05CH11231.

Publication
Shih, P., K. Vuu, N. Mansoori, L. Ayad, K. Louie, B. Bowen, T. Northen, and D. Loqué. 2016. “A Robust Gene-Stacking Method Utilizing Yeast Assembly for Plant Synthetic Biology,” Nature Communications 7(13215), DOI: 10.1038/ncomms13215. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


October 24, 2016

Metabolic Handoffs Among Microbial Community Members Drive Biogeochemical Cycles

Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system.

The Science
2,540 genomes that represent the majority of known bacterial phyla and 47 new phylum-level lineages were reconstructed from sediment and groundwater collected from a semi-arid floodplain near Rifle, CO. Analyses showed that inter-organism interactions are required to turn the carbon, sulfur and nitrogen biogeochemical cycles and revealed that complex patterns of community assembly are likely key to ecosystem functioning and resilience.

The Impact
The research almost doubled the number of major bacterial groups and provided detailed information about the ecosystem roles of organisms from these groups. The research dramatically increased understanding of subsurface biology and motivates new approaches to ecosystem modeling. The genomes represent a treasure-trove that will be mined for biotechnology.

Summary
The subterranean world hosts up to one fifth of all biomass, including microbial communities that drive transformations central to Earth’s biogeochemical cycles. However, little is known about how complex microbial communities in such environments are structured, and how inter-organism interactions shape ecosystem function. Terabase-scale cultivation-independent metagenomics was applied to aquifer sediments and groundwater and 2,540 high-quality near-complete and complete strain-resolved genomes that represent the majority of known bacterial phyla were constructed.  Some of these genomes derive from 47 newly discovered phylum-level lineages. Metabolic analyses spanning this vast phylogenetic diversity and representing up to 36% of organisms detected in the system were used to document the distribution of pathways in coexisting organisms. Consistent with prior findings indicating metabolic handoffs in simple consortia, it was shown that few organisms within the community conduct multiple sequential redox transformations. As environmental conditions change, different assemblages of organisms are selected for, altering linkages among the major biogeochemical cycles.

BER PM Contact
David Lesmes, SC-23.1, 301-903-2977

Contact
Susan Hubbard
Lawrence Berkeley National Laboratory
sshubbard@lbl.gov

Funding: This work was supported by Lawrence Berkeley National Laboratory’s Sustainable Systems Scientific Focus Area funded by the US Department of Energy, Office of Science, Office of Biological and Environmental Research.  Terabase-scale sequencing critical for this work was provided by the Joint Genome Institute via Community Science Program allocations.

Publication
K. Anantharaman, C. T. Brown, L. A. Hug, I.Sharon, C. J. Castelle, A. J. Probst, B. C. Thomas, A. Singh, M. J. Wilkins, U. Karaoz, E. L. Brodie, K. H. Williams, S. S. Hubbard, and J. F. Banfield. “Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system”. Nature Communications 7, ncomms13219 (2016). [DOI: 10.1038/ncomms13219]. (Reference link)

Topic Areas:

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



Tree showing all of bacterial diversity that is now represented by genomes, with the major lineages indicated by wedges. Research on the microbiology of the Rifle aquifer has provided new genomic information within previously identified groups (black wedges). In addition, many major bacterial groups were first identified and via study of the Rifle site (red and purple wedges). Red wedges indicate many major lineages that were first identified in the current study. Colored dots indicate the genomically predicted roles of members of these newly defined bacterial lineages in geochemical cycling. Remarkably, few major bacterial lineages have not been genomically sampled at this site (olive green wedges). [Image courtesy of Anantharaman et al. 2016. DOI: 10.1038/ncomms13219. Reprinted under CC by 4.0.]



October 13, 2016

Database of DNA Viruses and Retroviruses Debuts on Integrated Microbial Genomes Platform

The publicly accessible database promotes comparative analyses and ground-breaking discoveries through biological translation of sequence data.

The Science
A new database dedicated to global viral diversity has been developed by the Department of Energy Joint Genome Institute (DOE JGI). This database is the largest publicly available database for viruses, with 3,908 isolate reference DNA viruses and 264,413 computationally identified viral contigs from more than 6,000 ecologically diverse metagenomic samples. In a series of four articles recently published in Nucleic Acids Research, DOE JGI researchers also report on the latest updates to several publicly accessible databases and computational tools that benefit the global community of microbial researchers.

The Impact
Microbes play key roles in maintaining the planet’s biogeochemical cycles. Viruses, thought to outnumber microbes by 10-fold, exert major influences on microbial survival and community interactions. Advances in sequencing technologies have generated vast amounts of data about these viruses, requiring tools to manage and interpret the information. Recent updates focus on database analytical tools for microbial genomics and viruses relevant to DOE missions in bioenergy and environment.

Summary
Providing high-quality, publicly accessible sequence data goes hand-in-hand with developing and maintaining the databases and tools that the research community can harness to help answer scientific questions. In a recent series of articles published in Nucleic Acids Research, researchers at DOE JGI, a national scientific user facility, describe a database called Integrated Microbial Genomes with Virus Samples (IMG/VR). IMG/VR is a comprehensive computational platform integrating all the sequences in the database with associated metadata and analytical tools. IMG/VR follows on the heels of a recent DOE JGI viral diversity study report in Nature. Additional articles in the same issue describe updates to several publicly accessible, interactive databases since the last set of reports published in 2014. For example, as of July 2016, there were 47,516 archaeal, bacterial, and eukaryotic genomes in the IMG with Microbiome Samples (IMG/M) system, with researchers noting that number “represents an over 300% increase since September 2013.” IMG/M contains annotated DNA and RNA sequence data of archaeal, bacterial, eukaryotic, and viral genomes from cultured organisms; single cell genomes (SCG) and genomes from metagenomes from uncultured archaea, bacteria, and viruses; and metagenomes from environmental, host-associated, and engineered microbiome samples. Another paper concerns the Genomes OnLine Database (GOLD), a manually curated data management system that catalogs sequencing projects with associated metadata from around the world. In the current version of GOLD (v.6), all projects are organized based on a four-level classification system in the form of a study, organism (for isolates) or biosample (for environmental samples), sequencing project, and analysis project. A fourth paper focuses on the IMG Atlas of Biosynthetic gene Clusters (IMG-ABC). Launched in 2015, IMG-ABC enables researchers to search for biosynthetic gene clusters and secondary metabolites. Their latest update now incorporates ClusterScout, a tool for targeted identification of custom biosynthetic gene clusters across several thousand isolate microbial genomes, as well as a new search capability.

Contacts
Daniel Drell, Ph.D.
Program Manager
Biological Systems Science Division
Office of Biological and Environmental Research
Office of Science, U.S. Department of Energy
daniel.drell@science.doe.gov

Nikos Kyrpides
Prokaryote Super Program Head
DOE Joint Genome Institute
nckyrpides@lbl.gov

Funding
U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research
U.S. National Institutes of Health Data Analysis and Coordination Center

Publications

I.-M. A. Chen, et al., “IMG/M: Integrated genome and metagenome comparative data analysis system.” Nucleic Acids Research (2016). [DOI:10.1093/nar/gkw929] (Reference link)

S. Mukherjee, et al., “Genomes OnLine Database (GOLD) v.6: Data updates and feature enhancements.” Nucleic Acids Research (2016). [DOI: 10.1093/nar/gkw992] (Reference link)

D. Paez-Espino, et al., “IMG/VR: A database of cultured and uncultured DNA Viruses and retroviruses.” Nucleic Acids Research (2016). [DOI: 10.1093/nar/gkw1030] (Reference link)

M. Hadjithomas, et al., “IMG-ABC: New features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes.” Nucleic Acids Research (2016). [DOI: 10.1093/nar/gkw1103] (Reference link)

Related Links

IMG

GOLD

IMG/VR

IMG-ABC

JGI News Release: Unveiled: Earth's Viral Diversity

JGI Science Highlight: First Public Resource for Secondary Metabolites Searches

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Geographic distribution of biosamples and organisms encompassed by the Genomes OnLine Database. Organism location of isolation is marked with pink dots, biosample location with blue dots. [Image courtesy of Mukherjee et al., Nucleic Acids Research (2016). DOI: 10.1093/nar/gkw992]



October 12, 2016

Unraveling the Molecular Complexity of Cellular Machines and Environmental Processes

State-of-the-art mass spectrometer delivers unprecedented capability to users.

The Science
Two recent studies demonstrate the enormous potential for scientists to explore extremely complex molecular mixtures and systems frequently encountered in environmental, biological, atmospheric, and energy research.

The Impact
The Environmental Molecular Sciences Laboratory (EMSL), a Department of Energy Office of Science user facility, has an unprecedented ability to routinely analyze large intact proteins, precisely measure the fine structure of isotopes, and extract more information from complex natural organic matter mixtures. One of the world’s most powerful mass spectrometry instruments, a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T FTICR MS), is now available to the scientific community. Illustrating the power of this new instrument for biogeochemical research, EMSL scientists were able to make over 8,000 molecular formula assignments from dissolved organic matter mixtures using the 21T FTICR MS. In another study, EMSL users rapidly identified and discovered new types of metal-binding molecules known as siderophores, which are produced by bacterial cells.

Summary
As the highest-performance mass spectrometry technique, the FTICR MS has become increasingly valuable in recent years for various research applications. The FTICR MS determines the mass-to-charge ratio of ions by measuring the frequency at which ions rotate in a magnetic field, providing ultra-high resolution and mass measurement accuracy. The 21T FTICR MS, which is one of only two in the world with this high magnetic field strength, went online at EMSL in 2015. In a recent study, a team of EMSL scientists evaluated performance gains produced by this high magnetic field strength. They found this next-generation instrument empowers routine analysis of large intact proteins, precisely measures the fine structure of isotopes, and elicits more information than ever before from complex natural organic matter mixtures. The initial performance characterization of the 21T FTICR MS demonstrates enormous potential for future applications to extremely complex molecular mixtures and systems frequently encountered in environmental, biological, atmospheric, and energy research. Moreover, this unprecedented level of mass resolution and accuracy will help promote widespread use of top-down proteomics—an approach that enables accurate characterization of different protein variants with different biological activity. As a result, this transformative instrument will enable users from around the world to tackle previously intractable questions related to atmospheric, terrestrial, and subsurface processes; microbial communities; biofuel development; and environmental remediation.

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

PI Contact
Ljiljana Paša-Tolic
Environmental Molecular Sciences Laboratory
ljiljana.pasatolic@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, including support of the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility, and the "High Resolution and Mass Accuracy Capability" development project at EMSL.

Publications
J. B. Shaw, T.-Y. Lin, F. E Leach III, A. V. Tolmachev, N. Tolic, E. W. Robinson, D. W. Koppenaal, and L. Paša-Tolic, “21 Tesla Fourier transform ion cyclotron resonance mass spectrometer greatly expands mass spectrometry toolbox.” Journal of the American Society for Mass Spectrometry 27(12), 1929-36 (2016). DOI: 10.1007/s13361-016-1507-9. (Reference link)

L. R. Walker, M. M. Tfaily, J. B. Shaw, N. J. Hess, L. Pasa-Tolic, and D. W. Koppenaal, “Unambiguous identification and discovery of bacterial siderophores by direct injection 21 Tesla Fourier transform ion cyclotron resonance mass spectrometry.” Metallomics (2017). DOI: 10.1039/c6mt00201c. (Reference link)

Related Links
Unraveling Molecular Complexity of Natural Systems
Top-down Proteomics: Onward and Upward

Topic Areas:

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



The 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer will propel the future direction of environmental, biological, atmospheric, and energy research. [Image courtesy Pacific Northwest National Laboratory]



September 27, 2016

Oleaginous Yeasts Move One Step Closer to Becoming Industrial Biodiesel Producers

Engineering metabolic pathways and enzyme subcellular localization enables efficient production of fatty acids and other green chemicals.

The Science
Using a combination of different genetic engineering strategies, scientists were able to make oleaginous yeasts convert low-value carbon compounds into different fatty acids and alcohols that can be used for diesel-like fuel production and other industrial applications. The high levels of product achieved with this approach bring the development of a yeast biorefinery platform for high-value fuel and oleochemical production closer to reality.

The Impact
Oleaginous microorganisms, such as Yarrowia lipolytica, have great potential as industrial producers of biofuels and bioproducts due to their high lipid biosynthetic capacity. However, lipid metabolic engineering in eukaryotes is not advanced enough to take advantage of these organisms. This research demonstrates that a deeper understanding of different aspects of lipid metabolism, from genetic regulation to metabolic compartmentalization to enzyme structure, enables the design and engineering of new strains to substantially increase lipid production.  

Summary
Researchers at the Massachusetts Institute of Technology applied a multipronged strategy to engineer Y. lipolytica to produce several lipid molecules with applications as biofuels and other oleochemicals such as fatty acid ethyl esters, fatty alkanes, fatty acids, fatty alcohols, and triacylglycerides. This strategy included engineering Y. lipolytica lipid metabolism by expressing enzymes from other microorganisms within specific subcellular compartments within the yeast cells where specific lipids or their precursors are metabolized. Another approach was to engineer a chimeric enzyme to regulate the chain length of specific fatty acids. Finally, to increase the availability of acetyl-CoA building blocks for fatty acid synthesis, alternative acetyl-CoA pathways were designed to avoid the normal repression of acetyl-CoA synthesis by low nitrogen concentration in the medium. Production of different lipid molecules in these engineered strains was increased between 2 and 20 fold, paving the way toward developing industrial strains for commercial production of biodiesel and bioproducts from renewable sources.     

Contacts (BER PM)
Pablo Rabinowicz
Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
Gregory Stephanopoulos
Department of Chemical Engineering
Massachusetts Institute of Technology
Cambridge, MA
gregstep@mit.edu

Funding
This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science program (award DE-SC0008744).  

Publication
P. Xu, K. Qiao, W. S. Ahn, and G. Stephanopoulos, “Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals.” Proceedings of the National Academy of Sciences (USA) 113(39), 10848-853 (2016). [DOI: 10.1073/pnas.1607295113] (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Oleaginous yeasts such as Yarrowia lipolytica (pictured) can accumulate more than 80 percent of their dry weight as lipids, making them promising organisms for biodiesel production. [Image courtesy Massachusetts Institute of Technology/Peng Xu]



September 27, 2016

The Brown Rot Two-Step

Understanding the biomechanisms brown rot fungi use to degrade wood could lead to new tools for more efficient biofuel production. 

The Science
Wood’s complex structure of cellulose, long chains of linked sugar molecules, embedded in a scaffolding of polyaromatic lignin makes it highly resistant to biological or chemical decomposition. Brown rot fungi, however, possess a unique ability to attack the cellulose fraction of wood while avoiding the surrounding lignin. This study provides evidence that brown rot fungi accomplish this using a two-step process: (1) by secreting a set of chemicals and enzymes that open up the lignin framework, and then (2) releasing a second set of enzymes that break down the cellulose chains into sugars that are absorbed by the fungi.

The Impact
Understanding the newly discovered two-step mechanism of this degradation process could lead to the development of new biotechnology approaches for efficient and cost-effective conversion of wood cellulose into biofuels or bioproducts while leaving the lignin intact as a potential useful byproduct.

Summary
Wood-decomposing fungi are essential players in breaking down plant biomass in forest ecosystems and could provide important clues on how to more efficiently convert lignocellulose—the primary building block of wood cell walls—to biofuels and other products. Among these organisms, brown rot fungi are unique in their ability to selectively degrade the cellulose in wood while leaving the lignin portion mainly intact.To accomplish this task, these fungi generate highly reactive oxygen species that alter the chemical structure of wood and work in tandem with enzymes that break down cellulose chains. However, reactive oxygen species could just as easily damage the fungal enzymes as the wood structure, so researchers have long hypothesized that the fungi spatially segregate the oxidant generation process from the secreted enzymes using sets of chemical barriers. However, in this study, scientists found evidence that brown rot fungi separate the oxidants and enzymes in time rather than in space. This two-step wood decomposition mechanism was discovered by designing a simple, yet elegant experiment: brown rot fungi were grown in one direction along thin wood specimens separating the stages of wood decay linearly across the substrate. The wood was then cut into sections and analyzed for patterns of gene expression using whole-transcriptome shotgun sequencing (RNA-seq), assayed for relevant enzyme activity, and imaged using confocal and fluorescence microscopy. The researchers found that at early time points in the brown rot colonization, there was evidence of lignocellulose oxidation by reactive oxygen species and an increase in expression of genes important for plant cell wall-swelling. Both of these activities would weaken the structural integrity of wood and make it easier for enzymes to access cellulose chains. Only at later time points of colonization did brown rot fungi begin to produce glycoside hydrolase enzymes that break down cellulose chains into their component sugars. This unique fungal “pretreatment” strategy predates chemical pretreatment approaches used in industrial biomass processing by millions of years and could provide important new clues for improved conversion of woody plant materials into renewable cellulosic biofuels.

Contacts
(BER PM)
Dawn M. Adin
Program Manager, Office of Biological and Environmental Research
dawn.adin@science.doe.gov
Paul Bayer
Program Manager, Office of Biological and Environmental Research
paul.bayer@science.doe.gov

(PI Contact)
Jonathan S. Schilling
University of Minnesota
schillin@umn.edu  

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research under award numbers DE-SC0004012 (DOE Early Career Research) and DE-SC0012742. This research also used resources at the Environmental Molecular Sciences Laboratory, which is a DOE Office of Science user facility.

Publication
Zhang, J., G. N. Presley, K. E. Hammel, J.-S. Ryu, J. R. Menke, M. Figueroa, D. Hu, G. Orr, and J. S. Schilling. 2016. “Localizing Gene Regulation Reveals a Staggered Wood Decay Mechanism for the Brown Rot Fungus Postia placenta,” Proceedings of the National Academy of Sciences (USA) 113(39), 10968-973. DOI: 10.1073/pnas.1608454113. (Reference link)

Related Links
EMSL Highlight

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Image courtesy of Jonathan S. Schilling (University of Minnesota)
Pictured is a brown rot wood-degrading fungus Laetiporus sulphureus commonly known as the “chicken of the woods” fruiting on a hardwood log.



September 05, 2016

Microbial Metabolism Impacts Sustainability of Fracking Efforts

Through hydraulic fluids, surface microbes are colonizing the deep subsurface where they are adapting and thriving.

The Science
Hydraulic fracturing (“fracking”) is the industry standard for extracting hydrocarbons from shale formations, which provide one-third of natural gas energy resources worldwide. Poorly understood, however, are the biogeochemical changes that this process induces in the deep subsurface. In a recent study, researchers, for the first time, were able to reconstruct microbial genomes from shale formations that are being drilled for natural gas. Coupled with microbial metabolic information, the data shed considerable light on the impacts to microbial communities in the deep subsurface, as well as on the sustainability of energy extraction through this approach.

The Impact
Microbial metabolism and growth in hydrocarbon reservoirs are known to have both positive and negative impacts on energy recovery, but little is known about the structure, function, and activity of microorganisms in hydraulically fractured shale. This study provides evidence for microbial degradation of chemical additives and the potential for microbially induced corrosion and formation of biogenic methane. These findings could be used to develop strategies to reduce the risk of fracking-related environmental contamination and to improve long-term sustainability of energy extraction.

Summary
Hydraulic fracturing uses high-pressure injection of fresh water and chemical additives deep into the earth to generate extensive fractures in the shale matrix, thereby releasing hydrocarbons trapped in tiny pore spaces. A recent study—led by researchers from The Ohio State University, Department of Energy’s (DOE) Environmental Molecular Sciences Laboratory (EMSL), DOE Joint Genome Institute (JGI), and University of Maine—found that along with these fluids, microbes from the surface are also being injected and colonizing the deep subsurface, 2.5 km underground. To find out how this process may be impacting resident microbial community structure, function, and activity, the research team conducted metagenomic and metabolite analyses on input and produced fluids from gas wells for up to a year after hydraulic fracturing at two Appalachian basin shales: the Marcellus and Utica/Point Pleasant formations. The researchers used several nuclear magnetic resonance instruments at EMSL and high-throughput DNA sequencing technologies at JGI, both of which are DOE Office of Science user facilities. By reconstructing the first genomes of microbes in fractured shale, researchers discovered remarkable adaptations by microorganisms to survive the extreme chemical conditions produced by fracking. For example, microbes in fractured shales commonly consume injected chemical additives and produce an amino acid derivative called glycine betaine, which protects against high salinity by balancing the osmotic difference between the cell's surroundings and the internal cytoplasm. Glycine betaine is then taken up and used as a source of energy by other microbes, which, in turn, release metabolites that support methane-producing bacteria known to enhance energy recovery. On the other hand, salt-loving bacterial strains that synthesize glycine betaine also produce hydrogen sulfide, which contributes to equipment corrosion, risks environmental contamination, and decreases profits. Additional analysis revealed the majority of archaeal and bacterial genomes reconstructed from fluid samples showed evidence of acquired immunity against viruses, which actively infect other microbes vulnerable to fracking-related environmental stressors. Taken together, these findings illustrate the role of microbial communities resident in oil-bearing shales and begin to reveal a wide range of factors supporting long-term microbial persistence and adaptation to extreme environmental conditions in hydraulically fractured shales.

BER PM Contacts
Paul Bayer, SC-23.1, 301-903-5324
Dan Drell, SC-23.2, 301-903-4742

PI Contacts
Rebecca A. Daly
The Ohio State University
daly.130@osu.edu

David Hoyt
DOE Environmental Molecular Sciences Laboratory
david.hoyt@pnnl.gov

Susannah Tringe
DOE Joint Genome Institute
sgtringe@lbl.gov

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research (BER), and used resources at DOE’s Joint Genome Institute and Environmental Molecular Sciences Laboratory, which are DOE Office of Science user facilities. Both facilities are sponsored by BER and operated under contract numbers DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL). Additional funding was provided by the National Science Foundation’s Dimensions of Biodiversity (award number 1342701).

Publication
Daly, R. A., M. A. Borton, M. J. Wilkins, D. W. Hoyt, D. J. Kountz, R. A. Wolfe, S. A. Welch, D. N. Marcus, R. V. Trexler, J. D. MacRae, J. A. Krzycki, D. R. Cole, P. J. Mouser, and K. C. Wrighton. 2016. “Microbial Metabolisms in a 2.5-KM-Deep Ecosystem Created by Hydraulic Fracturing in Shales,” Nature Microbiology, DOI: 10.1038/nmicrobiol.2016.146. (Reference link)

Related Links
EMSL Highlight
JGI Highlight

Topic Areas:

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



Oil and gas well site in the Appalachian Basin similar to the well sites where researchers conducted metagenomic and metabolite analyses on hydraulic fluids. [Image courtesy of the Marcellus Shale Energy and Environment Laboratory]



September 01, 2016

Genomics Helps Advance Understanding of How an Important Bioenergy Feedstock Tolerates Environmental Stresses

First comprehensive study of an important protein family in the perennial woody plant Populus lays the foundation for functional characterization.

The Science
A genome-wide characterization of a family of plant-specific receptor proteins in the bioenergy feedstock Populus revealed tissue-specific expression and suggests a possible function in tolerance to environmental stresses.

The Impact
This comprehensive study of lectin receptor-like kinases (LecRLKs) in a woody plant provides the foundation for functional characterization of an important protein family.

Summary
Cell-surface receptor proteins play an important role in signal perception and processing, which, in turn, influence growth and development. The membrane-bound LecRLKs comprise a large family of such proteins. LecRLKs are specific to plants and are believed to be involved in responses to external stimuli such as pathogens and environmental stresses. Scientists with Oak Ridge National Laboratory’s Plant-Microbe Interface project report the first genome-wide analysis and classification of LecRLKs in the perennial woody model plant Populus, a bioenergy feedstock tree important for carbon sequestration, ecological systems studies, and biomass production. The researchers found that the LecRLK family was greatly expanded in Populus, with notably high levels of expression in the roots as compared with other plant tissues. They hypothesize that since the root system provides the interface for soil microbes, LecRLKs expressed in the roots may function to perceive microbial signals, which, in turn, influence plant health and tolerance of biotic and abiotic stresses. This first comprehensive study of LecRLKs in a woody plant lays the basis for functional characterization of an important protein family.

Contacts (BER PM)
Cathy Ronning 
SC-23.2
catherine.ronning@science.doe.gov

(PI Contact)
Jin-Gui Chen
Biosciences Division, Oak Ridge National Laboratory
chenj@ornl.gov

Funding
This work was supported by the Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science program, Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy [(DOE); DE-AC05-00OR22725], as well as DOE’s Joint Genome Institute, an Office of Science user facility (DE-AC02-05CH11231).

Publication
Yang, Y., J. Labbé, W. Muchero, X. Yang, S. S. Jawdy, M. Kennedy, J. Johnson, A. Sreedasyam, J. Schmutz, G. A. Tuskan, and J.-G. Chen. 2016. “Genome-Wide Analysis of Lectin Receptor-Like Kinases in Populus,” BMC Genomics 17, 699. DOI 10.1186/s12864-016-3026-2. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 23, 2016

Bacterial Protein Shows Promise for Efficiently Converting Plant Biomass to Biofuels

Enzyme has the highest known activity for hydrolyzing recalcitrant crystalline cellulose found in plant cell walls.

The Science
Glycoside hydrolases are microbial enzymes that play a key role in nutrient acquisition through the breakdown of cellulose—a major component of plant cell walls. A recent study showed that a protein from the bacterial glycoside hydrolase family 12 plays an unexpectedly important role in converting the hard-to-degrade crystalline form of cellulose and that it does so through a random mechanism unlike other hydrolases.

The Impact
The discovery of a glycoside hydrolase protein that is highly effective at breaking down rigid plant cell wall components could be harnessed to develop more efficient strategies for converting plant biomass to fuels and chemicals.

Summary
Microbes such as fungi and bacteria produce enzymes called glycoside hydrolases to acquire nutrients through the degradation of cellulose—carbohydrates that make up plant cell walls. Some of these enzymes are capable of breaking down the rigid, crystalline form of cellulose and, therefore, could be especially effective at efficiently converting tough plant biomass to fuels and chemicals. However, they have largely been studied in pure cultures of microorganisms, even though microorganisms break down cellulose as communities in the environment. To address this knowledge gap, a multi-institutional team of researchers led by scientists at the Department of Energy’s (DOE) Joint BioEnergy Institute (JBEI) combined comparative proteomics with biochemical measurements. They then assessed differences in glycoside hydrolases produced by diverse microbes in communities cultivated from green waste compost and grown on crystalline cellulose. The team used several mass spectrometry instruments at the Environmental Molecular Sciences Laboratory (EMSL) and high-throughput DNA sequencing technologies at the Joint Genome Institute, both of which are DOE Office of Science user facilities. Their analysis revealed that a glycoside hydrolase family 12 protein, produced by the bacterium Thermobispora bispora, plays a previously underappreciated important role in breaking down crystalline cellulose. The new findings suggest this protein could be especially effective at converting plant biomass to fuels and chemicals. More broadly, the study illustrates the power of comparative community proteomics to reveal novel insights into microbial proteins that could be harnessed for fuel production from renewable energy sources. This research represents collaboration among JBEI, Lawrence Berkeley National Laboratory, Sandia National Laboratories, Pacific Northwest National Laboratory, EMSL, and University of Applied Sciences Mannheim.

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

PI Contacts
Steven Singer
Lawrence Berkeley National Laboratory/Joint BioEnergy Institute
SWSinger@lbl.gov

Errol (Robby) Robinson
Pacific Northwest National Laboratory
errol.robinson@pnnl.gov

Funding
This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research (BER). Furthermore, this work was performed under the Facilities Integrating Collaborations for User Science (FICUS) initiative and used resources at DOE’s Environmental Molecular Sciences Laboratory and Joint Genome Institute, which are DOE Office of Science user facilities sponsored by BER.

Publication
J. Hiras, Y. W. Wu, K. Deng, C. D. Nicora, J. T. Aldrich, D. Frey, S. Kolinko, E. W. Robinson, J. M. Jacobs, P. D. Adams, T. R. Northen, B. A. Simmons, and S. W. Singer, “Comparative community proteomics demonstrates the unexpected importance of actinobacterial glycoside hydrolase family 12 protein for crystalline cellulose hydrolysis.” mBio 7(4), e01106-16 (2016). [DOI: 10.1128/mBio.01106-16]. (Reference link)

Related Links
Steve Singer bio
EMSL News

Topic Areas:

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



A glycoside hydrolase protein is highly effective at breaking down rigid plant cell wall components and could be used to develop more efficient strategies for converting plant biomass to fuels and chemicals. [Image courtesy Department of Energy Environmental Molecular Sciences Laboratory]



August 19, 2016

Advancing Toward Construction of a Bacterial Recoded Genome

Initial testing of a synthetic bacterial genome that uses 57 of the 64 natural codons showed minimal fitness impairment.

The Science
Taking advantage of the genetic code’s redundancy, a collaborative project led by researchers at Harvard University synthesized a bacterial genome that uses 57 of the 64 natural codons; the remaining seven codons were reassigned to nonstandard amino acids that can be used to develop novel protein functions. So far, DNA segments that span over 60% of the synthetic genome and contain over half the essential genes have been introduced into living cells to test for deleterious effects, and only very few cases showed significant growth defects.

The Impact
Reassigning several of the 64 natural codons in a bacterial genome enables the development of microbial strains with multiple combinations of proteins that can perform novel functions, while preventing the engineered strain from surviving if it escapes laboratory conditions. This large-scale, genome-wide recoding required developing design tools that must be fine-tuned after testing and gaining knowledge of rules that must be observed to synthesize functional genetic elements and networks. This research shows that recoding essential genes is possible and has uncovered fundamental design principles.    

Summary
To construct a completely recoded Escherichia coli genome, the researchers first used computational tools to design a genomic sequence lacking all instances of seven redundant codons and synthesized the genome in 87 fragments spanning 50 kb each. Testing of 55 of these fragments, which contain 63% of the genome and 52% of essential genes, showed that most of them caused limited or no change in growth and transcription levels. The recoded version of one gene resulted in severe fitness impairment, but the researchers were able to redesign the gene, allowing the strain to survive. At the same time, the researchers were able to optimize the design tools to further reduce potential growth defects in recoded microbes. This research demonstrates the feasibility of high-level recoding of microbial organisms to confer new functionality such as the development of new bioproducts. It also shows that genome-wide engineering approaches provide new knowledge on the fundamental principles that drive biological systems.  

Contacts (BER PM)
Pablo Rabinowicz
pablo.rabinowicz@science.doe.gov
(PI Contact)
George M. Church
Department of Genetics, Harvard Medical School
Wyss Institute for Biologically Inspired Engineering
Harvard University
Boston, MA
gchurch@genetics.med.harvard

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science (award DEFG02-02ER63445). Authors also acknowledge support from the U.S. Department of Defense and National Science Foundation.

Publication
Ostrov, N., M. Landon, M. Guell, G. Kuznetsov, J. Teramoto, N. Cervantes, M. Zhou, K. Singh, M. Napolitano, M. Moosburner, E. Shrock, B. Pruitt, N. Conway, D. Goodman, C. Gardner, G. Tyree, A. Gonzales, B. Wanner, J. Norville, M. Lajoie, and G. Church. 2016. “Design, Synthesis, and Testing Toward a 57-Codon Genome,” Science 353(6301), 819-22. DOI: 10.1126/science.aaf3639. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



By recoding bacterial genomes such as Escherichia coli (pictured), it is possible to create organisms that can potentially synthesize products not commonly found in nature. Image courtesy of iStock



August 02, 2016

Grasses Fight Drought by Squelching Root Growth

Shoot-borne roots that normally start developing in grasses weeks after germination are suppressed under drought conditions.  

The Science
Using fluorescent imaging technologies and direct observation of green foxtail roots excavated from soil, researchers discovered that root growth normally initiated from the crown (belowground shoot-root joint) is inhibited when water is scarce.  

The Impact
Drought tolerance is an important trait needed in bioenergy crops to enable their cultivation in marginal lands. As roots are the main conduit for water acquisition, understanding their biology is critical to discovering ways to improve bioenergy crops. The root system of potential bioenergy crops in the Poaceae family, such as switchgrass and sorghum, and the model grass Setaria viridis (green foxtail) is composed mostly by crown roots that emerge days or weeks after germination. However, little is known about their development under drought conditions. The discovery of a widespread mechanism of crown root suppression in grass species opens new avenues for improving bioenergy crop performance in dry environments.

Summary
A detailed study of root growth using traditional and new fluorescent imaging technologies in the model bioenergy crop Setaria showed that the crown (shoot-root node found belowground) senses the level of water conditions immediately surrounding the plant. At low soil humidity, root growth is arrested shortly after initiation, while root growth is rapidly resumed when water availability increases. Researchers from the Carnegie Institution for Science and international collaborators observed that drought-induced inhibition of root growth is also present in several other grasses, including the bioenergy crops sorghum and switchgrass and corn wild relatives, but not in highly domesticated corn lines. Furthermore, a corn mutant that lacks crown roots retains more water in the stem. These results suggest that grasses are adapted to inhibit root growth to preserve water and to induce crown root growth in response to precipitation to maximize water absorption in wet conditions. Genetic and transcriptomics analyses showed that oxidative-stress response genes may be involved in the process. The identification of the genes responsible for this phenomenon will be critical targets for engineering drought tolerance in bioenergy grasses.

Contacts (BER PM)
Pablo Rabinowicz
Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
José Dinneny
Department of Plant Biology
Carnegie Institution for Science, Stanford, CA 94305
jdinneny@carnegiescience.edu

Funding
This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under award DE-SC0008769. Additional support was provided by the National Science Foundation.

Publications
Sebastian, J., M. Yee, W. Viana, R. Rellán-Álvarez, M. Feldman, H. Priest, C. Trontin, T. Lee, H. Jiang, I. Baxter, T. Mockler, F. Hochholdinger, T. Brutnell, and J. Dinneny. 2016. “Grasses Suppress Shoot-Borne Roots to Conserve Water During Drought,” Proceedings of the National Academy of Sciences (USA) 113(31), 8861-66. DOI: 10.1073/pnas.1604021113. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


August 02, 2016

LuxR Homolog in a Cottonwood Tree Endophyte Activates Gene Expression in Response to Plant Signal or Specific Peptides

This new gene discovery opens the door for investigating other signals involved in plant-bacteria interactions.

The Science                       
Scientists discovered a gene in the bacterium Pseudomonas sp. GM79, a beneficial microbe commonly found within the roots of Populus trees (cottonwood), that is activated by signals exuded from the plant. 

The Impact
The study’s findings provide a model for investigating a possible new family of signals involved in plant-bacteria interactions that are present in dozens of bacterial species associated with economically important plants. 

Summary
Many beneficial soil bacteria are associated with plant roots, both outside the root (rhizosphere) and within (endophytic microbes). In Populus, a candidate bioenergy feedstock, the endophyte- and rhizosphere-associated communities are distinct, with a- and ?-Proteobacteria dominating the endophyte communities and Acidobacteria and a-Proteobacteria predominant within the rhizosphere. Proteobacteria isolated from Populus roots have been shown to possess acyl-homoserine lactone (AHL)-type quorum sensing (QS) activity, a cell-to-cell signaling system among bacteria that is dependent on cell density. The AHL QS system includes both signal synthases (encoded by luxI-type genes) and signal receptors (encoded by luxR-type genes), but some of the LuxR proteins have been found to respond instead to plant-derived chemical elicitors. Scientists at Oak Ridge National Laboratory, as part of the Plant-Microbe Interfaces Scientific Focus Area within the Department of Energy’s Office of Biological and Environmental Research, discovered a gene in a Proteobacteria Pseudomonas spGM79 isolated from Populus roots that is a plant signal-activated “orphan” member of the LuxR family of regulatory genes. The gene, pipR, is often flanked by predicted peptidase and peptide transporter genes and is closely related to a gene present in plant pathogens that similarly responds directly to plant-derived signals. Studies support the hypothesis that active transport of a peptide-like signal is required for the signal to interact with PipR, which then activates peptidase gene expression. The identification of a peptide ligand for PipR provides a foundation to identify plant-derived signals for orphan LuxR family proteins.

Contacts (BER PM)
Cathy Ronning
SC-23.2
catherine.ronning@science.doe.gov
(PI Contact)
Caroline Harwood
University of Washington, Seattle, WA
csh5@uw.edu

Funding
This work was funded by the Genomic Science program, Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy, as part of the Plant-Microbe Interfaces Scientific Focus Area (http://pmiweb.ornl.gov/).

Publications
Schaefer, A. L., Y. Oda, B. G. Coutinho, D. Pelletier, J. Weiburg, V. Venturi, E. P. Greenberg, and C. S. Harwood. 2016. “A LuxR Homolog in a Cottonwood Tree Endophyte that Activates Gene Expression in Response to a Plant Signal or Specific Peptides,” mBio 7(4), e01101-16. DOI: 10.1128/mBio.01101-16. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


July 19, 2016

Diverse Fungi Secrete Similar Suite of Decomposition Enzymes

Genomic and proteomic analyses reveal diversity in carbon turnover and other degradation processes.

The Science
Soil fungi secrete a wide range of enzymes that play an important role in biogeochemistry as well as in biofuel production and bioremediation of metal-contaminated soils and water. A recent study sheds new light on a suite of enzymes secreted by diverse fungal species commonly found in soil ecosystems worldwide.

The Impact
The findings reveal different fungal species secrete a rich set of enzymes that share similar functions, despite species-specific differences in the amino acid sequences of these enzymes. This information enhances understanding of the role fungi play in biogeochemical processes occurring in soil and could be used to engineer fungal enzymes for biofuel production and bioremediation efforts.

Summary
Fungi secrete a diverse repertoire of enzymes that break down tenacious plant material. These powerful enzymes degrade plant cell wall components such as cellulose and lignin, resulting in the release of carbon dioxide from soils with dead plant material into the atmosphere. As such, fungal enzymes are not only critical drivers of climate dynamics, but they also hold promise for cost-effective development of alternative transportation fuels. Moreover, the manganese [Mn(II)]-oxidizing capacity of certain fungal species can be harnessed to remove toxic metals from contaminated soils and water. Yet few studies have characterized enzymes secreted by diverse Mn(II)-oxidizing fungi that are commonly found in the environment. To address this knowledge gap, a team of researchers recently used liquid chromatography-tandem mass spectrometry (LC-MS/MS), genomic, and bioinformatic analyses to characterize and compare enzymes secreted by four Mn(II)-oxidizing Ascomycetes species. These four species were recently isolated from coal mine drainage treatment systems and a freshwater lake contaminated with high concentrations of metals and are associated with varied environments and common in soil ecosystems worldwide. The researchers performed LC-MS/MS-based comparative proteomics using the Linear Ion Trap Quadrupole Orbitrap Velos mass spectrometer at the Department of Energy’s (DOE) Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility. This analysis revealed that fungi secrete a rich yet functionally similar suite of enzymes, despite species-specific differences in the amino acid sequences of these enzymes. These findings enhance understanding of the role Ascomycetes species play in biogeochemistry and climate dynamics and reveal lignocellulose-degrading enzymes that potentially could be engineered for renewable energy production or bioremediation of metal-contaminated waters. This study represents a collaboration among scientists from Harvard University, EMSL, Pacific Northwest National Laboratory, Smithsonian Institution, DOE Joint Genome Institute (JGI), Centre National de la Recherche Scientifique and Aix-Marseille Université, King Abdulaziz University, University of Minnesota, and Woods Hole Oceanographic Institution.

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

PI Contact
Carolyn Zeiner
Harvard University
zeiner@bu.edu

Funding
This work was supported by DOE’s Office of Science, Office of Biological and Environmental Research, including support of EMSL and JGI, Office of Science user facilities, and Harvard University.

Publication
Zeiner, C. A., S. O. Purvine, E. M. Zink, L. PaÅ¡a-Tolić, D. L. Chaput, S. Haridas, S. Wu, K. LaButti, I. V. Grigoriev, B. Henrissat, C. M. Santelli, and C. M. Hansel. 2016. “Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi,” PLOS ONE 11(7), e0157844. [DOI:10.1371/journal.pone.0157844]. (Reference link)

Related Links
EMSL science highlight
JGI science highlight

Topic Areas:

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



Researchers compared fungal secretions to enhance understanding of the role Ascomycetes species play in soil biogeochemistry and climate dynamics. Image courtesy of the Department of Energy’s Environmental Molecular Sciences Laboratory



July 02, 2016

Unraveling the Complex Metabolism of a Potential Biofuels-Producing Green Alga

A genome-scale metabolic model of a green microalga is providing strategies for improving its growth.

The Science  
The green microalga, Chlorella vulgaris, has the potential to act as a cell factory in the production of biofuels and bioproducts. To better understand the complex and diverse metabolic capabilities of this green microalga, researchers transformed the organism’s genomic data into a mathematical model. This model enabled the researchers to understand and systematically analyze how the alga is able to grow in a variety of conditions including with just sunlight and carbon dioxide. The model then provided guidance on modifying the conditions to enhance growth performance.

The Impact
An in-depth understanding of how microorganisms use nutrients and grow is essential to improving the production of desired products, including biofuels and bioproducts. The developed model simulated different growth parameters simultaneously (e.g., nutritional resources, genetic modifications, and light source and availability) so that optimal conditions can be predicted. Optimizing growth conditions maximizes the probability of obtaining the desired experimental result, while also saving valuable time and resources.

Summary
The global movement toward more green-energy opportunities is resulting in the development of new approaches for producing renewable fuels in economical ways. The green microalga, C. vulgaris, is recognized as a promising candidate for biofuel production due to its ability to store high amounts of lipids and its natural metabolic versatility. However, many fundamental questions remain on how this alga and other microorganisms can more efficiently use nutritional sources not just for the organism’s growth, but also for sustainable and efficient production of biofuel and bioproducts. Researchers from the University of California, San Diego; Johns Hopkins University; University of Delaware; and National Renewable Energy Laboratory wanted to develop a way to more efficiently modify C. vulgaris to improve growth productivity. To do this, the scientists developed a compartmentalized genome-scale metabolic model that enabled quantitative insight into the organism’s metabolism. The model accurately predicted phenotypes under a variety of growth conditions including photoautotrophic, heterotrophic, and mixotrophic conditions. Model validation was performed using experimental data, laying the foundation for model-driven strain design and growth medium alteration to improve biomass yield. Model prediction of growth rates under various medium compositions and subsequent experimental tests showed an increased growth rate with the addition of the amino acids tryptophan and methionine. The reconstruction represents the most comprehensive model of eukaryotic photosynthetic organisms to date, based on genome size and number of genes in the reconstruction. With this metabolic model, researchers should be able to improve experimental design strategies for strain, production process, and final product yield optimization.

Contact (BER PM)
Dawn Adin, Ph.D.
Program Manager, Office of Biological and Environmental Research
dawn.adin@science.doe.gov  

Contacts (PIs)
Michael J. Betenbaugh
Department of Chemical and Biomolecular Engineering
Johns Hopkins University
beten@jhu.edu    

Karsten Zengler
Department of Bioengineering
University of California, San Diego
kzengler@ucsd.edu

Funding
This work was supported by the National Science Foundation (grant number 1332344); U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (grant number DE-SC0012658); and Mexican National Research Council (fellowship number 237897 to C.Z.).

Publication
C. Zuñiga, C.-T. Li, T.Huelsman, J. Levering, D. C. Zielinski, B. O. McConnell, C. P. Long, E. P Knoshaug, T. G. Guarnieri, M. R. Antoniewicz, M. J. Betenbaugh, and K. Zengler, “Genome-scale metabolic model for the green alga Chlorella vulgaris UTEX 395 accurately predicts phenotypes under autotrophic, heterotrophic, and mixotrophic growth conditions.” Plant Physiology 172, 589-602 (2016). [DOI: 10.1104/pp.16.00593] (Reference link)

Related Links
Zengler Laboratory Website
Betenbaugh Laboratory Website

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The polytrophic metabolism of photosynthetic microorganisms such as Chlorella vulgaris (pictured in test tubes) enables them to take advantage of different energy sources for growth. [Image courtesy of the National Renewable Energy Laboratory]



April 26, 2016

Poplar-Associated Bacterial Isolates Induce Additive Favorable Responses in a Constructed Plant-Microbiome System

These findings suggest microbiome phenotype can be predicted from phenotypes of individual community members.

The Science
A recent study showed that two species of plant growth-promoting bacteria enhanced beneficial plant traits such as root growth and photosynthetic potential in poplar trees, both by themselves and, in combination, in an additive manner.

The Impact
The effects observed in this constructed microbial community study suggest that microbiome function may be predicted in these systems from the additive functions of selected individual microbial species.

Summary
The diverse microbial communities that inhabit the zones within and surrounding the roots of plants, the “root microbiome,” have a significant influence on the host plant’s health and vitality. The root microbiome of Populus, a genus of trees that are a potential bioenergy feedstock, contains a high abundance of microbes known as β- and γ-Proteobacteria. Both of these classes include multiple bacterial species known to promote plant growth. To understand the contribution of individual microbiome members in a community, researchers at Oak Ridge National Laboratory (ORNL), funded by the Department of Energy’s (DOE) Plant-Microbe Interfaces Science Focus Area and U.S. Department of Agriculture-DOE Plant Feedstocks Genomics for Bioenergy program, studied a simplified community consisting of Pseudomonas (γ-Proteobacteria) and Burkholderia (β-Proteobacteria) bacterial strains inoculated on sterile Populus cuttings under controlled laboratory conditions. Alone and in combination, the two species increased root growth and photosynthetic potential and activated unique pathways relative to uninoculated controls.   Complementary data such as photosynthetic efficiency, gene expression, and metabolite expression data, in individual and in mixed inoculated treatments, indicate that the molecular effects of these bacterial strains are unique and additive. This work is the first constructed community study to show the additive host effects of bacteria, and the results suggest that microbiome function may be predicted from the synergistic effects of individual members of the microbial community.

Contacts
(BER PM)
Cathy Ronning, SC-23.2, catherine.ronning@science.doe.gov, 301-903-9549

(PI Contact)
Collin Timm
Biosciences Division, ORNL
timmcm@ornl.gov

Funding
This work was funded by DOE’s Office of Science, Office of Biological and Environmental Research, Biological Systems Science Division, Genomic Science and Plant Feedstock Genomics for Bioenergy programs (DE-SC0010423); and Plant-Microbe Interfaces Science Focus Area at Oak Ridge National Laboratory.

Publications
Timm, C. M., D. A. Pelletier, S. S. Jawdy, L. E. Gunter, J. A. Henning, N. Engle, J. Aufrecht, E. Gee, I. Nookaew, Z. Yang, T. Lu, T. J. Tschaplinksi, M. J. Doktycz, G. A. Tuskan, and D. J. Weston. 2016. “Two Poplar-Associated Bacterial Isolates Induce Additive Favorable Responses in a Constructed Plant-Microbiome System,” Frontiers in Plant Sciences 7:497. DOI: 10.3389/fpls.2016.00497. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


April 11, 2016

A New View of the Tree of Life

Access to a wealth of environments and the ability to reconstruct genomes for previously unknown and uncultured lineages has greatly expanded understanding of the diversity of life on earth.

The Science
A comprehensive three domain tree of life was constructed from all lineages for which sequenced genomes are available. The tree highlights the diversity contained in candidate phyla: lineages with no cultivated representatives for which genome sequences are derived from environmental surveys.

The Impact
The tree of life is one of the most important organizing principles in biology. The new depiction will be useful not only to biologists who study microbial ecology, but also to biochemists searching for novel genes and researchers studying evolution and earth history. This updated view highlights the weight of diversity found within the bacteria and within lineages with no cultured representatives.

Summary
This tree presents a new view of the diversity of life from a genome perspective. Exploration of new environments and deeper sequencing of well-studied systems continue to uncover new organisms and lineages on the tree. To construct a comprehensive tree of life, researchers gathered 3,085 genomes representing all genera for which genomes are available and including over 1,000 newly reconstructed genomes targeting candidate phyla representatives. Sample sites for new genomes included extreme environments like Chile’s Atacama Desert salt flats and Yellowstone National Park hot springs, but also more common environments such as groundwater, estuarine sediment, meadow soil, and dolphin oral microbiomes. The tree inferred from this genomic perspective shows the predominance of bacterial diversity compared to the divergence seen in the Archaea and Eukarya.  Collapsing the tree based on sequence divergence rather than taxonomy highlighted the amount of diversity found within candidate phyla, emphasizing the importance of environmental surveys for discovery of organisms not tractable in laboratory experiments.

Contacts (BER PM)
Todd Anderson
Todd.Anderson@science.doe.gov

David Lesmes
David.Lesmes@science.doi.gov

(PI Contact)
Jillian Banfield
University of California Berkeley
jbanfield@berkeley.edu

Funding
This research was largely supported by Lawrence Berkeley National Laboratory’s (LBNL) Genomes to Watershed Scientific Focus Area funded by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research (BER) under contract DE-AC02-05CH11231. Additional support was provided by LBNL EFRC award DE-AC02-05CH11231; National Aeronautics and Space Administration NESSF grant 12 PLANET12R-0025 and National Science Foundation DEB grant 1406956; DOE BER grant DOE-SC10010566; Office of Naval Research grants N00014-07-1-0287, N00014-10-1-0233, and N00014-11-1-0918; and the Thomas C. and Joan M. Merigan Endowment at Stanford University. In addition, funding was provided by the Ministry of Economy, Trade, and Industry of Japan, and metagenome sequence was generated by DOE’s Joint Genome Institute via the Community Science Program.

Publication
Hug, L. A., B. J. Baker, K. Anantharaman, C. T. Brown, A. J. Probst, C. J. Castelle, C. N. Butterfield, A. W. Hernsdorf, Y. Amano, K. Ise, Y. Suzuki, N. Dudek, D. A. Relman, K. M. Finstad, R. Amundson, B. C. Thomas, and J. F. Banfield. 2016. “A New View of the Tree of Life,” Nature Microbiology 1(16048), DOI: 10.1038/nmicrobiol.2016.48. (Reference link)

Topic Areas:

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


April 04, 2016

A One-Pot Recipe for Making Jet Fuel

Researchers use engineered bacteria to simplify biofuels production, potentially lowering cost.

The Science
Researchers isolated an Escherichia coli mutant that tolerates a liquid salt used to break apart plant biomass into sugary polymers. Because the salt solvent, known as ionic liquid (IL), interferes with the later stages of biofuels production, it has to be removed before proceeding, a process that requires time and money. The researchers genetically engineered the E. coli strain to excrete an IL-tolerant cellulase and used the resulting sugars to synthesize d-limonene, a jet fuel precursor.

The Impact
IL-tolerant bacteria enable a “one-pot” method for producing advanced biofuels from a slurry of pretreated plant material, helping to streamline the production process, which is critical to making biofuels a viable competitor with fossil fuels.

Summary
Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multistep process. Each of the steps—deconstruction of the cellulose, hemicellulose, and lignin that are bound together in the plant cell wall; addition of enzymes to release sugars; and conversion into the desired biofuel—is done in separate pots. Significant effort has gone into developing efficient solutions to these discrete steps, but few studies report the consolidation of the multistep workflow into a single pot reactor system. Researchers at the Department of Energy’s (DOE) Joint BioEnergy Institute (JBEI) demonstrate a one-pot biofuel production process that uses an IL (1-ethyl-3-methylimidazolium acetate) for pretreating switchgrass biomass. This IL is highly effective in deconstructing lignocellulose, but leaves behind a residue that is toxic to standard cellulase and the microbial production host. JBEI scientists established that an amino acid mutation in the gene rcdA leads to an E. coli strain that is highly tolerant to ILs. To develop a strain for a one-pot process, they engineered this IL-tolerant strain to express a d-limonene production pathway. The JBEI researchers also screened previously reported IL-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the IL-tolerant E. coli strain to secrete this IL- tolerant cellulase. The final strain was found to digest pretreated biomass and use the liberated sugars to produce the jet fuel candidate precursor d-limonene in a one-pot process.

Contacts (BER PM)
N. Kent Peters
Program Manager, Office of Biological and Environmental Research
kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
Aindrila Mukhopadhyay
Joint BioEnergy Institute, Emeryville, CA, USA
amukhopadhyay@lbl.gov

Funding
This work was part of the Joint BioEnergy Institute supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research through contract DE-AC02-05CH11231.

Publication
Frederix, M., et al. 2016. “Development of an E. coli strain for One-Pot Biofuel Production from Ionic Liquid Pretreated Cellulose and Switchgrass,” Green Chemistry, DOI: 10.1039/c6gc00642f. (Reference link)

Related Links
News release

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Researchers in a microbiology lab at the Joint BioEnergy Institute are working to streamline the biofuels production process. Image courtesy Lawrence Berkeley National Laboratory



March 30, 2016

Engineering Intracellular Organelles to Increase Production of Useful Chemicals by Confining Their Metabolic Pathways

Scientists move closer to engineering metabolic pathways within yeast intracellular compartments tailored for desired purposes.

The Science
Engineering microbes to produce valuable bioproducts or biofuels is often complicated, because the product is deleterious to the cell or the chemical conditions in the cytosol are not favorable for the required chemical reactions. To overcome this challenge, researchers at the University of California (UC), Berkeley, have developed a system to facilitate the introduction of enzymes into the yeast peroxisome, isolating the selected metabolic pathway from the cytosolic environment.

The Impact
The development of a versatile intracellular organelle to confine engineered metabolic pathways will facilitate metabolic engineering. This research advances the repurposing of the yeast peroxisome to isolate synthetic metabolic pathways that would be inefficient if expressed dissolved in the cytosol. This system will make it possible to engineer eukaryotic cells to produce high yields of useful chemicals that cannot be achieved in traditional biological systems.

Summary
High-yield production of bioproducts and fuels in microbial systems requires metabolic flux to be directed toward an engineered pathway. However, this redirection of metabolic flux is difficult to achieve because cells tend to divert metabolic flux toward native cellular processes. Engineered metabolic pathways have been confined to organelles such as the mitochondrion or the vacuole to isolate them from the host's metabolism, but the cell needs those organelles for its normal functions and, therefore, they cannot be completely repurposed. On the other hand, yeast can live without peroxisomes, making this an ideal organelle to isolate newly designed metabolic pathways and their products. A research team at UC Berkeley has discovered a protein signal that allows the efficient targeting of engineered proteins into the peroxisome. The researchers also devised a high-throughput method to measure the efficiency of the process and demonstrated the feasibility of the approach by introducing a simple metabolic pathway that produces a colored compound into the yeast peroxisome. The strategy can now be used to sequester useful metabolic pathways into the peroxisome to produce high yields of valuable chemicals and fuels.

Contacts (BER PM)
Pablo Rabinowicz
Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
John Dueber
Bioengineering Department
University of California, Berkeley
jdueber@berkeley.edu

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science under Early Career Research Program award DE-SC0008084. Authors also acknowledge support from the National Science Foundation and U.S. Department of Defense.

Publications
DeLoache, W. C., Z. N. Russ, and J. E. Dueber. 2016. “Towards Repurposing the Yeast Peroxisome for Compartmentalizing Heterologous Metabolic Pathways,” Nature Communications 7, 11152. DOI: 10.1038/ncomms11152. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 25, 2016

Improving Lipid Yields for Biofuel Production

New insights into lipid metabolism in yeast could benefit biofuel production.

The Science
Using a comprehensive system-wide approach, researchers identified regulators of metabolic pathways that drive lipid accumulation in a genetically tractable yeast species.

The Impact
A better understanding of the metabolic pathways that regulate lipid accumulation in yeast could be harnessed to improve lipid yields and enhance the efficiency of biofuel production.

Summary
The yeast Yarrowia lipolytica is capable of accumulating a large amount of lipids when nitrogen is limited. This ability, along with its amenability to genetic methods, has made Y. lipolytica an attractive model for generating high-value lipids for biofuel production. However, relatively little is known about the factors that regulate enzymatic pathways responsible for lipid accumulation in this species. To address this knowledge gap, a team of researchers from Pacific Northwest National Laboratory (PNNL) integrated metabolome, proteome, and phosphoproteome data to characterize lipid accumulation in response to limited nitrogen in Y. lipolytica. The researchers used a microscopy system that integrates nonlinear two-photon excitation, laser-scanning confocal microscopy, and fluorescence lifetime imaging at the Environmental Molecular Sciences Laboratory (EMSL), a U.S. Department of Energy (DOE) scientific user facility. In this first global study of protein phosphorylation in Y. lipolytica, the researchers focused their analysis on changes in the expression and phosphorylation state of regulatory proteins, including kinases, phosphatases, and transcription factors. They found that lipid accumulation in response to nitrogen limitation results from two distinct processes: (1) higher production of malonyl-CoA from excess citrate increases the pool of building blocks for lipid production, and (2) decreased capacity for β-oxidation reduces lipid consumption. These findings provide new genetic targets that could be manipulated to improve lipid yields in future metabolic engineering efforts.

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

(PI Contact)
Scott E. Baker
EMSL
Scott.Baker@pnnl.gov
509-372-4759

Funding
This work was supported by DOE’s Office of Science, Office of Biological and Environmental Research (BER), including support of EMSL, an Office of Science user facility; BER Genomic Science program; William Wiley Distinguished Postdoctoral Fellowship; and BER-funded Pan-omics program at PNNL.

Publication
Pomraning, K. R., Y.-M. Kim, C. D. Nicora, R. K. Chu, E. L. Bredeweg, S. O. Purvine, D. Hu, T. O. Metz, and S. E. Baker. 2016. “Multi-Omics Analysis Reveals Regulators of the Response to Nitrogen Limitation in Yarrowia lipolytica,” BMC Genomics 17(138). DOI: 10.1186/s12864-016-2471-2. (Reference link).

Topic Areas:

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


February 18, 2016

Biofuel Tech Straight from the Farm

Herbivore digestion of lignocellulosic biomass involves a large variety of enzymes.

The Science
Herbivores eat many types of lignocellulosic plants, and fungi digest this material in the animals’ guts. A new study has characterized several fungi involved in this digestion process, identifying a large number of enzymes that work synergistically to degrade the raw biomass.

The Impact
Industry is exploring strategies to more effectively turn biomass like wood and grasses into fuel or chemicals. Because the matrix of complex molecules found in plant cell walls—lignin, cellulose, and hemicellulose—is difficult to break down using biological methods, costly pretreatments with heat or chemicals are necessary. The discovery of new, highly effective biomass-degrading enzymes in anaerobic fungi could accelerate the development of a process to convert lignocellulose feedstocks into fermentable sugars without pretreatment, potentially leading to more efficient conversion of raw biomass to biofuels and biobased products.

Summary
Scientists have long known that anaerobic fungi living in the guts of herbivores play a significant role in helping those animals digest plants. However, culturing these fungi in the lab is difficult because they cannot survive in the presence of oxygen and must be grown in sealed containers. A research team led by Michelle O’Malley at the University of California, Santa Barbara, isolated three species of these fungi in feces from goats, horses, and sheep. The enzymes expressed by these fungi work together to break down crude, untreated plant biomass. The research showed that the fungi adapt their enzymes to the different kinds of plant materials eaten by these animals, so that wood, grass, or agricultural waste all can be efficiently digested. Each of the fungi studied was found to contribute in a characteristic way, tailoring their combined action to the particular type of biomass being digested. These findings could help in identifying distinctive enzymes from other anaerobic gut fungi, with potential applications for biomass processing and sustainable biofuel production.

Contacts
PM Contact
Pablo Rabinowicz
Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

PI Contact
Michelle A. O’Malley
Department of Chemical Engineering
University of California, Santa Barbara
momalley@engineering.ucsb.edu

Funding
This work was supported by the Office of Biological and Environmental Research (BER) within the U.S. Department of Energy’s (DOE) Office of Science under Early Career Research Program award DE-SC0010352. A portion of this research was performed under the JGI-EMSL Collaborative Science Initiative and used resources at DOE’s Joint Genome Institute (JGI) and Environmental Molecular Sciences Laboratory (EMSL), which are DOE Office of Science user facilities and sponsored by BER. Authors also acknowledge support from the U.S. Department of Agriculture (Award 2011-67017-20459) and Institute for Collaborative Biotechnologies through grant W911NF-09-0001.

Publications
Solomon, K., et al. "Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes." Science (2016). [DOI: 10.1126/science.aad1431].   (Reference link

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



Microbes found in the digestive tract of large herbivores offer attractive enzyme platforms for lignocellulosic processing. [Image courtesy of iStock]



February 08, 2016

New Real-Time Approach for Monitoring Chemical Production by Genetically Engineered Microbes

Fluorescent sensors were developed to measure the productivity of bacteria engineered to synthesize precursors for plastics and other materials.  

The Science      
Metabolic engineering of microbes has great potential for sustainable and environmentally friendly production of industrial chemicals. Researchers at Harvard University have designed molecular tools (sensors) that enable them to follow the production of precursors for plastics and other chemicals in engineered microorganisms. These sensors produce increasing fluorescence as the amount of the desired product augments (i.e., "sensing" the presence of the product), making it possible to rapidly select the genetic modifications that result in the highest chemical yields.

The Impact
Constructing new microorganisms that make high amounts of desired compounds requires designing, modifying, and testing many different strains to ultimately select the best producer. Those tests use laborious and costly analytical techniques. The fluorescent sensors developed by this research enable rapid detection of individual strains that produce the largest amounts of desired chemicals by just measuring fluorescence. Coupled with cell sorting technologies, these sensors will enable the testing of millions of engineered strains in a single day.      
 
Summary
This research has resulted in the development of a genetic sensor that provides a fluorescent readout proportional to the intracellular concentration of 3-hydroxypropionate, a valuable plastic precursor also called 3HP. This sensor required the introduction of several enzymes into the model bacterium Escherichia coli to convert 3HP into acrylate (another plastic precursor). Next, the gene for a fluorescent reporter whose expression is activated by acrylate also was introduced into the same E. coli strain so that when acrylate is produced, fluorescence can be detected and used as proxy for the amount of 3HP synthesized. With this system, the researchers could easily identify a strain and culture conditions that produced over 20 times more 3HP than previously achieved. At the same time, this research demonstrated the first heterologous pathway for microbial production of acrylate. The investigators proved the flexibility of the approach by designing a similar sensor to monitor muconate (used to make nylon) and glucarate (needed for manufacturing detergents and other chemicals). The fluorescent biosensors developed by this research combined with fluorescence-based cell sorting will accelerate the development of sustainable production of relevant chemicals such as biofuels and biopolymers in engineered microbial systems.

Contacts (BER PM)
Pablo Rabinowicz
Office of Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

(PI Contact)
George M. Church
Wyss Institute for Biologically Inspired Engineering
Harvard University
Boston, MA
gchurch@genetics.med.harvard

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science award DEFG02-02ER63445. Authors also acknowledge support from the National Science Foundation.  

Publications
Rogers, J. K., and G. M. Church. 2016. “Genetically Encoded Sensors Enable Real-Time Observation of Metabolite Production,” Proceedings of the National Academy of Sciences (USA) 113(9), 2388-93. DOI: 10.1073/pnas.1600375113. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


February 05, 2016

New Understanding of One of Nature’s Best Biocatalysts for Biofuels Production

Discovery of a new enzyme system sheds further light on a microbe’s ability to efficiently break down inedible plant matter for conversion to biofuels and biobased chemicals.

The Science
Researchers found that Clostridium thermocellum—an anaerobic, thermophilic microorganism—uses a previously unknown mechanism to degrade cellulose (scaffolded cellulase enzymes not attached to the bacterial cell wall), in addition to other known degradation mechanisms (cellulosomes and free enzymes).

The Impact
This discovery helps explain C. thermocellum’s superior ability to digest biomass and demonstrates the highly diverse strategies evolved in nature for biomass conversion. Researchers are using the study’s findings to develop optimal systems for breaking down lignocellulosic biomass to produce biofuels and biobased chemicals.

Summary
Lignocellulosic biomass is the largest source of organic matter on Earth, making it a promising renewable feedstock for producing biofuels and chemicals. Currently, however, the main bottleneck in biofuel production is the low efficiency of cellulose conversion, which leads to high production costs. To date, C. thermocellum is the most efficient microorganism known for solubilizing lignocellulosic biomass. Its high cellulose digestion capability has been attributed to the organism’s efficient cellulases consisting of both a free enzyme system and a tethered cellulosomal system, wherein multiple carbohydrate active enzymes are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. U.S. Department of Energy (DOE) BioEnergy Science Center (BESC) researchers recently discovered that C. thermocellum also expresses a type of cellulosomal system that is not bound to the cell wall, a “cell-free” cellulosomal system. Researchers believe the cell-free cellulosome complex functions as a “long-range” cellulosome because it can diffuse away from the cell and degrade polysaccharide substrates distant from the bacterial cells. This discovery reveals that C. thermocellum utilizes not only all the previously known cellulase degradation mechanisms (cellulosomes and free enzymes), but also a new category of scaffolded enzymes not attached to the cell. This unexpected finding explains C. thermocellum’s superior performance on biomass, demonstrating that nature’s strategies for biomass conversion are not yet fully understood and could provide further opportunities for microbial enzyme discovery and engineering efforts.

Contacts (BER PM)
N. Kent Peters
Program Manager, Office of Biological and Environmental Research
kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
Yannick Bomble
Research Scientist, Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, and BESC, Oak Ridge National Laboratory, Oak Ridge, Tennessee
yannick.bomble@nrel.gov

Funding
This work was supported by BESC, a DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research within DOE's Office of Science. A portion of this work also was supported by the United States-Israel Binational Science Foundation, Jerusalem, Israel; Israel Science Foundation, Israeli Center of Research Excellence; European Union NMP.2013.1.1-2: CellulosomePlus Project 8 number 604530; and the ERA-IB Consortium (EIB.12.022) FiberFuel. 

Publications
Xu, Q., et al. 2016. “Dramatic Performance of Clostridium thermocellum Explained by Its Wide Range of Cellulase Modalities,” Science Advances 2(2), e1501254. DOI: 10.1126/sciadv.1501254. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER



The microbe Clostridium thermocellum (stained green) is seen growing on poplar tissue. [Image courtesy of Oak Ridge National Laboratory]



January 12, 2016

Using Bacteria to Achieve High Solubilization of Biomass with Minimal Pretreatment

Thermophilic bacteria prove to be efficient biocatalysts for biomass solubilization.

The Science
A comprehensive comparison of lignocellulosic solubilization by various thermophilic bacteria to standard enzyme treatment found microbial solubilization of cellulosic biomass to be more effective, and enhanced by mechanical disruption.

The Impact
Using thermophilic bacteria instead of expensive yeast enzymes to decompose biomass into its sugars for fermentation into biofuels will greatly reduce costs and potentially simplify the process.

Summary
Feedstock recalcitrance is the greatest barrier to cost-effective production of cellulosic biofuels. To overcome this recalcitrance, existing commercial cellulosic ethanol facilities employ thermochemical pretreatment with subsequent addition of fungal cellulase. However, processing cellulosic biomass without thermochemical pretreatment may be possible using thermophilic, cellulolytic bacteria. Researchers at the Department of Energy’s (DOE) BioEnergy Science Center (BESC) examined the ability of various thermophilic bacteria to solubilize autoclaved, but otherwise unpretreated cellulosic biomass. Carbohydrate solubilization of mid-season harvested switchgrass after 5 days ranged from 24 percent to 65 percent, with Clostridium thermocellum showing the best results among the four thermophiles tested. This finding was as much as fivefold better than with the standard method using a fungal cellulase cocktail and yeast fermentation. Other findings showed that there was equal fractional solubilization of glucan and xylan, and, importantly, that there was no biological solubilization of the noncarbohydrate fraction of biomass. A fivefold improvement over standard treatment was observed when using the most effective biocatalyst. Using thermophilic bacteria in biomass-solubilizing systems may enable a reduction in the amount of nonbiological processing required and, in particular, substitution of cotreatment for pretreatment.

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

(PI Contact)
Lee Lynd
Professor, Thayer School of Engineering, Dartmouth College
lee.lynd@dartmouth.edu

Funding
This research was sponsored by BESC, a DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research within DOE's Office of Science. TYN was supported by the National Science Foundation. The generation of the CCRC series of plant cell wall glycan-directed monoclonal antibodies used was supported by NSF's Plant Genome Program.

Publication
Paye, J. M. D., et al. 2016. “Biological Lignocellulose Solubilization: Comparative Evaluation of Biocatalysts and Enhancement via Cotreatment,” Biotechnology for Biofuels 9(8), DOI 10.1186/s13068-015-0412-y. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 07, 2016

A Novel Lipid Pathway Makes Massive Quantity of Surface Wax on Bayberry Fruit

Pathway gives metabolic engineers new tools for producing high-value lipids.

The Science
Bayberry fruits produce the highest amount of surface wax known in nature. Recent biochemical and gene expression data have revealed a novel biosynthesis pathway for the waxy layer that is more closely related to cutin biosynthesis than conventional triacylglyceride biosynthesis.

The Impact
The discovery of how the Bayberry fruit produces massive amounts of unique surface wax aids in understanding the plant’s mechanism for producing and secreting nonmembrane glycerolipids and suggests ways to engineer pathways for high-value waxy lipid production in biomass crops.

Summary
Scientists from the Department of Energy’s (DOE) Great Lakes Bioenergy Research Center (GLBRC) studied how Bayberry fruits accumulate massive quantities of a unique surface wax with a structure similar to triacylglycerol seed oils. Research on plants that produce such large amounts of surface lipids is providing insights into the molecular features and biochemical pathways for plant lipid secretion and thus may help in developing strategies to engineer lipid production in non-seed tissues. The GLBRC scientists examined changes in fruit anatomy and details of the chemical structures secreted by Bayberry fruits, and quantified the accumulation of wax through fruit development. Biochemical pathway analysis by [14C]-labeling and transcript analysis by RNA-seq revealed features of Bayberry wax accumulation that are distinctly different from conventional triacylglycerol production. Together, these results indicate that the extracellular glycerolipids in Bayberry wax are synthesized by a novel pathway that differs from previously defined triacylglycerol biosynthesis pathways. An increased understanding of this process may prove useful in engineering plants for secretion of high-energy and high-value lipids, particularly those that have toxic or negative consequences when accumulated inside cells.

Contacts
(BER PM)

N. Kent Peters, SC-23.2, kent.peters@science.doe.gov, 301-903-5549

(PI Contact)
John B. Ohlrogge
Michigan State University
ohlrogge@msu.edu

Funding
This work was funded by GLBRC (DOE, Office of Science, Office of Biological and Environmental Research DE-FC02-07ER64494) and a National Science and Engineering Research Council of Canada postgraduate fellowship (PGS-D3).

Publications
Simpson, J. P., and J. B. Ohlrogge. 2016. “A Novel Pathway for Triacylglycerol Biosynthesis Is Responsible for the Accumulation of Massive Quantities of Glycerolipids in the Surface Wax of Bayberry (Myrica pensylvanica) Fruit, The Plant Cell 28(1), 248–64. DOI: 10.1105/tpc.15.00900. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 04, 2016

Increased Production of Bioplastics in Engineered Bacteria

Metabolic engineering doubles the production of ethylene in Escherichia coli.

The Science
Researchers analyzed growth media and nutrient supplements to identify candidate genes that affect yields in an engineered E. coli strain that produces ethylene, a hydrocarbon used in the production of a wide range of chemicals and plastics. Guided by the results of those analyses, the researchers further engineered the bacterial strain, altering several metabolic and regulatory genes to more than double the original ethylene production levels.

The Impact
Ethylene is currently derived from fossil fuels through an energy-intensive process called steam cracking. The production of plastics and many other products and chemicals creates huge demand for ethylene, so biological production of ethylene has great potential to reduce the industry's carbon footprint. Ethylene biosynthesis has been engineered in microbial systems, but with low yields. The identification and engineering of selected E. coli metabolic and regulatory genes in this research has resulted in a substantial increase in ethylene yield, advancing the sustainable bioproduction of this critical hydrocarbon.

Summary
Ethylene is one of the most industrially important chemicals derived from petroleum. Therefore, scientists have been trying to develop biological systems to produce ethylene in a sustainable way. Expression of a heterologous bacterial ethylene-forming enzyme (EFE) in E. coli has resulted in the production of ethylene, but the yields were too low for industrial purposes. Researchers at the National Renewable Energy Laboratory and University of Colorado Boulder conducted a study of the effects of different nutrients and substrates present in the growth medium for the EFE-expressing E. coli strain to be able to predict which genes significantly affect ethylene yields. Guided by those findings, they re-engineered E. coli to minimize competing pathways within central metabolism and to overproduce key enzymes predicted to increase ethylene productivity. The re-engineered strain produced more than twice as much ethylene relative to the original EFE-expressing E. coli strain. Those yields can be further improved by identifying and engineering additional enzymes and regulatory factors that prevent higher metabolic flow toward ethylene biosynthesis. This work advances the development of a sustainable ethylene production industry that is not dependent on fossil fuels.  

Contacts (BER PM)
Pablo Rabinowicz, SC-23.2, pablo.rabinowicz@science.doe.gov, 301-903-0379

(PI Contact)
Pin–Ching Maness
National Renewable Energy Laboratory, Golden, Colorado
pinching.maness@nrel.gov

Funding
This work was supported by the Office of Biological and Environmental Research within the U.S. Department of Energy’s Office of Science under award DE-SC008812.

Publications
Lynch, S., C. Eckert, J. Yu, R. Gill, and P. C. Maness. 2016. “Overcoming Substrate Limitations for Improved Production of Ethylene in E. coli,” Biotechnology for Biofuels 9:3. DOI: 10.1186/s13068-015-0413-x. (Reference link)

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER