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News Article | May 26, 2017
Site: www.eurekalert.org

One of the biggest barriers in the commercial production of sustainable biofuels is to cost-effectively break down the bioenergy crops into sugars that can then be converted into fuel. To reduce this barrier, bioenergy researchers are looking to nature and the estimated 1.5 million species of fungi that, collectively, can break down almost any substance on earth, including plant biomass. As reported May 26, 2017 in Nature Microbiology, a team led by researchers at the University of California (UC), Santa Barbara has found for the first time that early lineages of fungi can form complexes of enzymes capable of degrading plant biomass. By consolidating these enzymes, in effect into protein assembly lines, they can team up to work more efficiently than they would as individuals. The work was enabled by harnessing the capabilities of two U.S. Department of Energy (DOE) Office of Science User Facilities, the DOE Joint Genome Institute (JGI) at Lawrence Berkeley National Laboratory (Berkeley Lab) and the Environmental Molecular Sciences Laboratory (EMSL) at Pacific Northwest National Laboratory (PNNL). "There are protein complexes in bacteria called cellulosomes that pack together the enzymes to break down plant biomass," said study senior author Michelle O'Malley of UC Santa Barbara. "The idea is that these clusters are better at attacking biomass because they are keeping the different enzymes in place with plugs called dockerins so they work more efficiently. This has been detailed in bacteria for more than 20 years, but now seen for the first time in fungi." With help from both the DOE JGI and EMSL, the team has now found protein complexes in anaerobic gut fungi that O'Malley said in principle do the same thing--attack plant biomass as a cluster of enzymes. While they found that many of enzymes in these complexes resulted from horizontal gene transfers with gut bacteria, they also noted differences in the composition compared to the bacterial cellulosomes. For one thing, both dockerins and scaffoldin are not similar between fungi and bacteria. Also, the bacterial cellulosomes are species-specific. Think of them as the high school clique that does everything together. In contrast, the fungal structures that appear analogous to the bacterial cellulosomes are like the high school kids who could easily move among various social groups and are comprised of clusters of enzymes that can "dock" and work in other fungi. The study involved a comparative genomics analyses of five fungi that belong to the Neocallimastigomycetes, a clade of the early-diverging lineages that are not well-studied. Three of the fungi were isolated from animal gut samples collected by the UC Santa Barbara team and sequenced and annotated by the DOE JGI. "Proteomics data and genomics data enabled us to figure out what these complexes are and go hunting for them in other genomes," O'Malley said. "The three genomes are really well resolved to the point where you can start looking at what's there, what's regulating enzyme production, and how enzymes have evolved." Study co-senior author and DOE JGI Fungal Genomics Program head Igor Grigoriev noted that a multi-omics approach that harnessed the genomics and molecular characterization capabilities through a collaborative science initiative allowing researchers to access multiple user facilities in one proposed project known as Facilities Integrating Collaborations for User Science (FICUS), was critical for the research. "It's the first time we've seen parts of the fungal cellulosome," Grigoriev said. "Through the JGI-EMSL FICUS initiative, proteomics allowed us to find the first of these really large ~700 kiloDaltons (kDa) fungal proteins that hold all enzymes together (compared to the molecular weight of 34 kDa of an average protein). Then the high quality of genome assemblies enabled identification of multiple copies of this protein in each of the gut fungi genomes. Just having proteomics or sequencing tools isn't enough since these proteins are not similar to anything else outside of Neocallimastigomycetes. Though the fungal cellulosome was discovered through proteomics, we needed genomics and transcriptomics to decode all its parts." The work is an extension of O'Malley's studies of anaerobic gut fungi, which appeared in Science last year (watch her talk from the 2016 DOE JGI Genomics of Energy & Environment Meeting at bit.ly/JGI2016OMalley). It's a lot of the same players, but we're digging deeper now because we have high-resolution genomes, and we didn't have them then," she said. "We're able to conduct more comparative genetics and now we're trying to figure out the ecological roles in their microbiome." Understanding in greater detail the protein mechanisms of biomass degradation is crucial to advancing DOE's agenda to develop sustainable biofuels from plant feedstocks. High throughput sequencing, combined with sophisticated proteomics, illuminates not just the diversity and complexity of fungal biomass degradation capacities but also furnishes a knowledge basis for exploitation of these abilities with synthetic biology and metabolic engineering approaches. Support for this work was provided by the DOE Office of Science, including an Early Career Research Program award, as well as the National Science Foundation, the U.S. Department of Agriculture, the U.S. Army Research Office, and the University of California. The U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility at Lawrence Berkeley National Laboratory, is committed to advancing genomics in support of DOE missions related to clean energy generation and environmental characterization and cleanup. DOE JGI, headquartered in Walnut Creek, Calif., provides integrated high-throughput sequencing and computational analysis that enable systems-based scientific approaches to these challenges. Follow @doe_jgi on Twitter. EMSL, the Environmental Molecular Sciences Laboratory, is a DOE Office of Science User Facility. Located at Pacific Northwest National Laboratory in Richland, Wash., EMSL offers an open, collaborative environment for scientific discovery to researchers around the world. Its integrated computational and experimental resources enable researchers to realize important scientific insights and create new technologies. Follow EMSL on Facebook, LinkedIn and Twitter. Interdisciplinary teams at Pacific Northwest National Laboratory address many of America's most pressing issues in energy, the environment and national security through advances in basic and applied science. Founded in 1965, PNNL employs 4,400 staff and has an annual budget of nearly $1 billion. It is managed by Battelle for the U.S. Department of Energy's Office of Science. As the single largest supporter of basic research in the physical sciences in the United States, the Office of Science is working to address some of the most pressing challenges of our time. For more information on PNNL, visit the PNNL News Center, or follow PNNL on Facebook, Google+, LinkedIn and Twitter. DOE's Office of Science is the largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit science.energy.gov.


News Article | May 26, 2017
Site: phys.org

As reported May 26, 2017 in Nature Microbiology, a team led by researchers at the University of California (UC), Santa Barbara has found for the first time that early lineages of fungi can form complexes of enzymes capable of degrading plant biomass. By consolidating these enzymes, in effect into protein assembly lines, they can team up to work more efficiently than they would as individuals. The work was enabled by harnessing the capabilities of two U.S. Department of Energy (DOE) Office of Science User Facilities, the DOE Joint Genome Institute (JGI) at Lawrence Berkeley National Laboratory (Berkeley Lab) and the Environmental Molecular Sciences Laboratory (EMSL) at Pacific Northwest National Laboratory (PNNL). "There are protein complexes in bacteria called cellulosomes that pack together the enzymes to break down plant biomass," said study senior author Michelle O'Malley of UC Santa Barbara. "The idea is that these clusters are better at attacking biomass because they are keeping the different enzymes in place with plugs called dockerins so they work more efficiently. This has been detailed in bacteria for more than 20 years, but now seen for the first time in fungi." With help from both the DOE JGI and EMSL, the team has now found protein complexes in anaerobic gut fungi that O'Malley said in principle do the same thing—attack plant biomass as a cluster of enzymes. While they found that many of enzymes in these complexes resulted from horizontal gene transfers with gut bacteria, they also noted differences in the composition compared to the bacterial cellulosomes. For one thing, both dockerins and scaffoldin are not similar between fungi and bacteria. Also, the bacterial cellulosomes are species-specific. Think of them as the high school clique that does everything together. In contrast, the fungal structures that appear analogous to the bacterial cellulosomes are like the high school kids who could easily move among various social groups and are comprised of clusters of enzymes that can "dock" and work in other fungi. The study involved a comparative genomics analyses of five fungi that belong to the Neocallimastigomycetes, a clade of the early-diverging lineages that are not well-studied. Three of the fungi were isolated from animal gut samples collected by the UC Santa Barbara team and sequenced and annotated by the DOE JGI. "Proteomics data and genomics data enabled us to figure out what these complexes are and go hunting for them in other genomes," O'Malley said. "The three genomes are really well resolved to the point where you can start looking at what's there, what's regulating enzyme production, and how enzymes have evolved." Study co-senior author and DOE JGI Fungal Genomics Program head Igor Grigoriev noted that a multi-omics approach that harnessed the genomics and molecular characterization capabilities through a collaborative science initiative allowing researchers to access multiple user facilities in one proposed project known as Facilities Integrating Collaborations for User Science (FICUS), was critical for the research. "It's the first time we've seen parts of the fungal cellulosome," Grigoriev said. "Through the JGI-EMSL FICUS initiative, proteomics allowed us to find the first of these really large ~700 kiloDaltons (kDa) fungal proteins that hold all enzymes together (compared to the molecular weight of 34 kDa of an average protein). Then the high quality of genome assemblies enabled identification of multiple copies of this protein in each of the gut fungi genomes. Just having proteomics or sequencing tools isn't enough since these proteins are not similar to anything else outside of Neocallimastigomycetes. Though the fungal cellulosome was discovered through proteomics, we needed genomics and transcriptomics to decode all its parts." The work is an extension of O'Malley's studies of anaerobic gut fungi, which appeared in Science last year (watch her talk from the 2016 DOE JGI Genomics of Energy & Environment Meeting at bit.ly/JGI2016OMalley). It's a lot of the same players, but we're digging deeper now because we have high-resolution genomes, and we didn't have them then," she said. "We're able to conduct more comparative genetics and now we're trying to figure out the ecological roles in their microbiome." Understanding in greater detail the protein mechanisms of biomass degradation is crucial to advancing DOE's agenda to develop sustainable biofuels from plant feedstocks. High throughput sequencing, combined with sophisticated proteomics, illuminates not just the diversity and complexity of fungal biomass degradation capacities but also furnishes a knowledge basis for exploitation of these abilities with synthetic biology and metabolic engineering approaches. Explore further: Finding a new major gene expression regulator in fungi More information: Charles H. Haitjema et al, A parts list for fungal cellulosomes revealed by comparative genomics, Nature Microbiology (2017). DOI: 10.1038/nmicrobiol.2017.87


PubMed | Michigan State University, University of Queensland, American Type Culture Collection, Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures and 3 more.
Type: Journal Article | Journal: Standards in genomic sciences | Year: 2014

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project with the objective of sequencing 250 bacterial and archaeal genomes. The two major goals of that project were (a) to test the hypothesis that there are many benefits to the use the phylogenetic diversity of organisms in the tree of life as a primary criterion for generating their genome sequence and (b) to develop the necessary framework, technology and organization for large-scale sequencing of microbial isolate genomes. While the GEBA pilot project has not yet been entirely completed, both of the original goals have already been successfully accomplished, leading the way for the next phase of the project. Here we propose taking the GEBA project to the next level, by generating high quality draft genomes for 1,000 bacterial and archaeal strains. This represents a combined 16-fold increase in both scale and speed as compared to the GEBA pilot project (250 isolate genomes in 4+ years). We will follow a similar approach for organism selection and sequencing prioritization as was done for the GEBA pilot project (i.e. phylogenetic novelty, availability and growth of cultures of type strains and DNA extraction capability), focusing on type strains as this ensures reproducibility of our results and provides the strongest linkage between genome sequences and other knowledge about each strain. In turn, this project will constitute a pilot phase of a larger effort that will target the genome sequences of all available type strains of the Bacteria and Archaea.


News Article | January 27, 2016
Site: phys.org

In a study published January 27, 2016 in Nature Communications, a team led by researchers at the DOE Joint Genome Institute (JGI), a DOE Office of Science User Facility, utilized the largest collection of metagenomic datasets to uncover a completely novel bacterial phylum that they have dubbed "Kryptonia." "We were interested in looking for novel, divergent bacterial or archaeal sequences that hadn't been previously characterized," said study first author Emiley Eloe-Fadrosh, a DOE JGI research scientist. "We didn't have a particular target to go after, but reasoned that there was likely a wealth of untapped diversity just waiting to be discovered in all the metagenomic data." A researcher analyzing vast quantities of genomic data is not unlike a beachcomber slowly scanning a beach with a metal detector. Both are searching for a signal in the noise that indicates buried treasure, be it novel microbes or pirate gold. The team started with 5.2 trillion bases (Terabases or Tb) of sequence in the Integrated Microbial Genomes with Microbiome Samples (IMG/M) system. After scouring this equivalent of over 1,700 human genomes or 1 million E. coli bacterial genomes, the team identified long sequences that contained a phylogenetic marker (DNA corresponding to ribosomal RNA, rRNA) commonly used to assign all life (bacteria, archaea, and eukaryotes) into a particular classification system. The team identified sequences from four different geothermal springs - Great Boiling Spring, Nevada, Dewar Creek Spring in Canada, and the Gongxiaoshe and Jinze pools in China - that could not be placed into any recognizable phylum. Reconstructing the genomes from metagenomic datasets and single cell genomes yielded four lineages belonging to the novel candidate phylum, named Kryptonia (Candidatus Kryptonia) from the Greek word for "hidden." Given that there are currently 35 cultured bacterial and archaeal phyla, and roughly the same number of recognized uncultured phyla, Eloe-Fadrosh says the identification of a novel candidate phylum was a surprise. "It's not every day that you find a completely new phylum. With all the studies that have been conducted in hot springs, there's an assumption that all novelty has been found. But we found these unknown lineages in high abundance." The analysis of the nearly complete Kryptonia genomes recovered from this metagenomics dataset also revealed the presence of the CRISPR-Cas phage defense system in these organisms. Using this information, the team was able to track the global biogeographic distribution of Kryptonia vis-à-vis the putative phages infecting the bacteria. "While a lot of research and media attention is gathering around the biotechnological applications of the CRISPR-Cas system, we are very excited about using it as a powerful tool in reconstructing the infection history of the organisms, as well as a fingerprint to uncover and trace the correlated viruses," said Prokaryote Super Program Head Nikos Kyrpides, a co-author of the paper. Analyses of Kryptonia reveal that the bacteria need to rely on other microbes for several nutritional requirements, suggesting a reason this candidate phylum had not been found previously despite its abundance in geothermal springs. "We hypothesize that Kryptonia engages in a metabolic partnership, and it's very challenging to cultivate bacteria that have unique interactions in the wild that can't necessarily be replicated in the lab," said Eloe-Fadrosh, who credited the combined power of metagenomics and single-cell genomics to capture the novel microbes described in the paper. "I think one of the grand challenges for the field is to quantify microbial diversity, and these technologies are getting us closer to making that a reality." The work reinforces the perspective published in Science last year by DOE JGI Director Eddy Rubin and Microbial Program head Tanja Woyke. "There are reasons to believe that current approaches may indeed miss taxa, particularly if they are very different from those that have so far been characterized," they wrote. "Past explorations of available metagenomic data sets have focused on the discovery of matches to the known genes and genomes—an analysis that is naturally biased against uncovering completely novel life." Eloe-Fadrosh said the team found unique metabolic pathways in Kryptonia, hints that there may be other novel enzymes related to biological pathways waiting to be uncovered. "Just like Taq polymerase was revolutionary to molecular biology, there could be an enzyme in Kryptonia with biotechnological relevance," she added. Her words echo the speculations offered by study co-author and DOE JGI collaborator Brian Hedlund of the University of Nevada, Las Vegas. Noting that Kryptonia play a role in lignocellulose degradation, he added that there are potential resources still waiting to be tapped for biotechnology applications in the "microbial dark matter" from which Kryptonia has only just emerged. For example, he said, companies are marketing enzymes from thermophiles for quick diagnostic tests including those from previous research enabled by the DOE JGI on Yellowstone hot pools. "I do believe that applications are there if people spend time and money looking for the microbes," he said. Eloe-Fadrosh spoke about the search for Kryptonia at the DOE JGI 2015 Genomics of Energy and Environment Meeting. Watch the video below: Explore further: Microbial genomes help propose phylum name


News Article | August 29, 2016
Site: phys.org

The study enhances understanding of the role fungi play in processes occurring in soil. The study could be used to engineer fungal enzymes for biofuel production and bioremediation efforts. 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 containing 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 from biomass. 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. Recently, a team of researchers used liquid chromatography-tandem mass spectrometry (LC-MS/MS), genomic analyses, and bioinformatic analyses to characterize and compare enzymes secreted by four Mn(II)-oxidizing Ascomycetes species. These four species were 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 Environmental Molecular Sciences Laboratory (EMSL), a Department of Energy's (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 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. Explore further: Researchers annotate genome of the smallest known fungal plant pathogen More information: Carolyn A. Zeiner et al. Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi, PLOS ONE (2016). DOI: 10.1371/journal.pone.0157844


News Article | November 17, 2016
Site: phys.org

Now a team from the Max-Planck-Institute (MPI) for Terrestrial Microbiology in Marburg, Germany, by tapping the DNA synthesis expertise of the U.S. Department of Energy Joint Genome Institute (DOE JGI) has reverse engineered a biosynthetic pathway for more effective carbon fixation. This novel pathway is based on a new CO2-fixing enzyme that is nearly 20 times faster than the most prevalent enzyme in nature responsible for capturing CO2 in plants by using sunlight as energy. "We had seen how efforts to directly assemble synthetic pathways for CO2-fixation in a living organism did not succeed so far," said Tobias Erb of MPI, who led the study. "So we took a radically different, reductionist approach by assembling synthetic principal components in a bottom-up fashion in a test tube." The team started with several theoretical CO2-fixation routes that could result in continuous carbon cycling. But they didn't stop there. "We did not restrict our design efforts to known enzymes, but considered all reactions that seemed biochemically feasible," Erb said. Unlike DNA sequencing, where the language of life is read from the genome of an organism, DNA synthesis entails first the identification of a particular genetic element - such as an enzyme for fixing carbon from the atmosphere—and writing and expressing that code in a new system. In the end, they sourced, through sequencing and synthesis, 17 different enzymes from 9 different organisms across the three kingdoms of life and orchestrated these parts to achieve a proof of principle CO2-fixation pathway performance that exceeds that which can be found in nature. Erb calls this the "CETCH cycle" for crotonyl-CoA/ethylmalonyl-12 CoA/hydroxybutyryl-CoA. Because it 'cetches' CO2 more efficiently from the atmosphere. By deploying the concept of metabolic "retrosynthesis," dismantling the reaction step by step all the way back to smaller precursors, the team juggled the thermodynamic conditions and came up with a strategy that yielded more promising results that competed favorably with natural-occurring metabolic pathways. Then they plumbed the depths of the public databases for enzymes that would support their model and selected several dozen to try out. "We first reconstituted its central CO2-fixation reaction sequence stepwise, providing the ingredients to catalyze all the desired reactions. Then, by following the flux of CO2 we discovered which particular key reaction was rate-limiting." This turned out to be methylsuccinyl-CoA dehydrogenase (Mcd), part of a family of enzymes involved in respiration ¬- the metabolic reaction in the cells of organisms to convert nutrients like carbon into units of energy. "To overcome this limitation, we engineered the Mcd to use oxygen as an electron acceptor, to amp up the function, but this was not quite enough," said Erb. "We had to replace the original pathway design with alternative reaction sequences, used further enzyme engineering to minimize side reactions of promiscuous enzymes, and introduced proofreading enzymes to correct for the formation of dead-end metabolites," Erb said. In support of the MPI team's efforts, the DOE JGI synthesized hundreds of Enoyl-CoA Carboxylase/Reductase (ECR) enzyme variants through its Community Science Program. This enabled the MPI team to zero in on the ECR with the highest CO2-fixation activity to successfully build a more efficient artificial CO2 fixation pathway in a test tube. "ECRs are supercharged enzymes that are capable of fixing CO2 at the rate of nearly 20 times faster than the most widely prevalent CO2-fixing enzyme in nature, RuBisCo, which carries out the heavy lifting involved in photosynthesis," Erb said. This chemical process harnesses sunlight to turn carbon dioxide into sugars that cells can use as energy along with other natural processes on the planet and accounts for the transformation of some 350 billion tons of CO2 annually. Seventy years ago this phenomenon captured the imagination of early Berkeley Lab researcher Melvin Calvin who, along with Andrew Benson and James Bassham, described, in plants, algae and microorganisms, the cycle that now bears their names, and for which Calvin was awarded the Nobel Prize in 1961. This generation of researchers are concerned about how to capture excess carbon dioxide, remove it from the atmosphere and render it into energy and natural products for the economy. "Now Berkeley Lab through the DOE Joint Genome Institute, has been a major contributor to our understanding of the vast genetic diversity of microorganisms and their roles in the environment, particularly in carbon cycling," said Yasuo Yoshikuni, the head of the DNA Synthesis Science group at the DOE JGI. "By sequencing underexplored phyla from ecologically important niches, we have homed in on the genes and pathways that we now are able to synthesize in the lab to unravel novel strategies that nature uses for carbon metabolism. Identifying these genes encoding CO2 -fixing enzymes and their biological function, is one of the important missing pieces in the climate puzzle." Emboldened by the successful reconstitution of a synthetic enzymatic network in a test tube for the conversion of CO2 into organic products that is superior to chemical processes and competes with favorably with those in nature, Erb said this opens the door for other future applications. "These could include the introduction of synthetic CO2-fixation cycles into organisms to bolster natural photosynthesis, or say, in combination with photovoltaics, lead the way to artificial photosynthesis, this might at the end jumpstart the design of self-sustaining, completely synthetic carbon metabolism in bacterial and algal systems." Yoshikuni looks to a future where DNA sequencing and biological functions further converge leveraging DNA synthesis. "Through DOE JGI's high-throughput sequencing capabilities coupled with the rapidly decreasing price of DNA synthesis, we continue to enable our user community in bringing to light the physiological potential of microorganisms and microbial communities. In the longer term, we hope to expect to see these test-tube results yield a new generation of real bioproducts delivered to address critical energy and environmental challenges." The broader significance of this work is to dramatically illustrate the increased role of "engineering thinking" in biotechnology, as the accelerated characterization of the biological "parts list" emerging from high throughput genome sequencing furnishes greater opportunities to reconstruct by design capacities in living organisms that address DOE mission needs in bioenergy and environment. Explore further: Eating air, making fuel: Scientists engineer bacteria to create sugar from the greenhouse gas carbon dioxide More information: "A synthetic pathway for the fixation of carbon dioxide in vitro," Science, science.sciencemag.org/cgi/doi/10.1126/science.aah5237


News Article | November 17, 2016
Site: www.eurekalert.org

Despite the vast diversity of organisms on the planet that express enzymes for the conversion of carbon dioxide into such organic compounds as sugars - as plants do through photosynthesis - the efforts to harness these capabilities to transform CO2 into high-value products such as biofuel and renewable chemicals have met with limited success. While increasing concentration of CO2 in the atmosphere poses a challenge, researchers also see it as an opportunity. Now a team from the Max-Planck-Institute (MPI) for Terrestrial Microbiology in Marburg, Germany, by tapping the DNA synthesis expertise of the U.S. Department of Energy Joint Genome Institute (DOE JGI) has reverse engineered a biosynthetic pathway for more effective carbon fixation. This novel pathway is based on a new CO2-fixing enzyme that is nearly 20 times faster than the most prevalent enzyme in nature responsible for capturing CO2 in plants by using sunlight as energy. "We had seen how efforts to directly assemble synthetic pathways for CO2-fixation in a living organism did not succeed so far," said Tobias Erb of MPI, who led the study. "So we took a radically different, reductionist approach by assembling synthetic principal components in a bottom-up fashion in a test tube." The team started with several theoretical CO2-fixation routes that could result in continuous carbon cycling. But they didn't stop there. "We did not restrict our design efforts to known enzymes, but considered all reactions that seemed biochemically feasible," Erb said. Unlike DNA sequencing, where the language of life is read from the genome of an organism, DNA synthesis entails first the identification of a particular genetic element - such as an enzyme for fixing carbon from the atmosphere ¬- and writing and expressing that code in a new system. In the end, they sourced, through sequencing and synthesis, 17 different enzymes from 9 different organisms across the three kingdoms of life and orchestrated these parts to achieve a proof of principle CO2-fixation pathway performance that exceeds that which can be found in nature. Erb calls this the "CETCH cycle" for crotonyl-CoA/ethylmalonyl-12 CoA/hydroxybutyryl-CoA. Because it 'cetches' CO2 more efficiently from the atmosphere. By deploying the concept of metabolic "retrosynthesis," dismantling the reaction step by step all the way back to smaller precursors, the team juggled the thermodynamic conditions and came up with a strategy that yielded more promising results that competed favorably with natural-occurring metabolic pathways. Then they plumbed the depths of the public databases for enzymes that would support their model and selected several dozen to try out. "We first reconstituted its central CO2-fixation reaction sequence stepwise, providing the ingredients to catalyze all the desired reactions. Then, by following the flux of CO2 we discovered which particular key reaction was rate-limiting." This turned out to be methylsuccinyl-CoA dehydrogenase (Mcd), part of a family of enzymes involved in respiration ¬- the metabolic reaction in the cells of organisms to convert nutrients like carbon into units of energy. "To overcome this limitation, we engineered the Mcd to use oxygen as an electron acceptor, to amp up the function, but this was not quite enough," said Erb. "We had to replace the original pathway design with alternative reaction sequences, used further enzyme engineering to minimize side reactions of promiscuous enzymes, and introduced proofreading enzymes to correct for the formation of dead-end metabolites," Erb said. In support of the MPI team's efforts, the DOE JGI synthesized hundreds of Enoyl-CoA Carboxylase/Reductase (ECR) enzyme variants through its Community Science Program (see below). This enabled the MPI team to zero in on the ECR with the highest CO2-fixation activity to successfully build a more efficient artificial CO2 fixation pathway in a test tube. "ECRs are supercharged enzymes that are capable of fixing CO2 at the rate of nearly 20 times faster than the most widely prevalent CO2-fixing enzyme in nature, RuBisCo, which carries out the heavy lifting involved in photosynthesis," Erb said. This chemical process harnesses sunlight to turn carbon dioxide into sugars that cells can use as energy along with other natural processes on the planet and accounts for the transformation of some 350 billion tons of CO2 annually. Seventy years ago this phenomenon captured the imagination of early Berkeley Lab researcher Melvin Calvin who, along with Andrew Benson and James Bassham, described, in plants, algae and microorganisms, the cycle that now bears their names, and for which Calvin was awarded the Nobel Prize in 1961. This generation of researchers are concerned about how to capture excess carbon dioxide, remove it from the atmosphere and render it into energy and natural products for the economy. "Now Berkeley Lab through the DOE Joint Genome Institute, has been a major contributor to our understanding of the vast genetic diversity of microorganisms and their roles in the environment, particularly in carbon cycling," said Yasuo Yoshikuni, the head of the DNA Synthesis Science group at the DOE JGI. "By sequencing underexplored phyla from ecologically important niches, we have homed in on the genes and pathways that we now are able to synthesize in the lab to unravel novel strategies that nature uses for carbon metabolism. Identifying these genes encoding CO2 -fixing enzymes and their biological function, is one of the important missing pieces in the climate puzzle." Emboldened by the successful reconstitution of a synthetic enzymatic network in a test tube for the conversion of CO2 into organic products that is superior to chemical processes and competes with favorably with those in nature, Erb said this opens the door for other future applications. "These could include the introduction of synthetic CO2-fixation cycles into organisms to bolster natural photosynthesis, or say, in combination with photovoltaics, lead the way to artificial photosynthesis, this might at the end jumpstart the design of self-sustaining, completely synthetic carbon metabolism in bacterial and algal systems." Yoshikuni looks to a future where DNA sequencing and biological functions further converge leveraging DNA synthesis. "Through DOE JGI's high-throughput sequencing capabilities coupled with the rapidly decreasing price of DNA synthesis, we continue to enable our user community in bringing to light the physiological potential of microorganisms and microbial communities. In the longer term, we hope to expect to see these test-tube results yield a new generation of real bioproducts delivered to address critical energy and environmental challenges." The broader significance of this work is to dramatically illustrate the increased role of "engineering thinking" in biotechnology, as the accelerated characterization of the biological "parts list" emerging from high throughput genome sequencing furnishes greater opportunities to reconstruct by design capacities in living organisms that address DOE mission needs in bioenergy and environment. This research was supported by the European Research Council, the Swiss National Science Foundation, ETH Zurich and the Max-Planck Society. DOE JGI is a DOE Office of Science User Facility. The DOE Joint Genome Institute, through its Community Science Program, offers access to DNA synthesis resources. The next deadline falls on January 30, 2017. For more information, see: http://jgi. . The U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility at Lawrence Berkeley National Laboratory, is committed to advancing genomics in support of DOE missions related to clean energy generation and environmental characterization and cleanup. DOE JGI, headquartered in Walnut Creek, Calif., provides integrated high-throughput sequencing and computational analysis that enable systems-based scientific approaches to these challenges. Follow @doe_jgi on Twitter. DOE's Office of Science is the largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit science.energy.gov.


News Article | November 17, 2016
Site: www.sciencedaily.com

Despite the vast diversity of organisms on the planet that express enzymes for the conversion of carbon dioxide into such organic compounds as sugars -- as plants do through photosynthesis -- the efforts to harness these capabilities to transform CO into high-value products such as biofuel and renewable chemicals have met with limited success. While increasing concentration of CO in the atmosphere poses a challenge, researchers also see it as an opportunity. Now a team from the Max-Planck-Institute (MPI) for Terrestrial Microbiology in Marburg, Germany, by tapping the DNA synthesis expertise of the U.S. Department of Energy Joint Genome Institute (DOE JGI), has reverse engineered a biosynthetic pathway for more effective carbon fixation. This novel pathway is based on a new CO -fixing enzyme that is nearly 20 times faster than the most prevalent enzyme in nature responsible for capturing CO in plants by using sunlight as energy. The study was published in the November 18, 2016 issue of Science. "We had seen how efforts to directly assemble synthetic pathways for CO -fixation in a living organism did not succeed so far," said Tobias Erb of MPI, who led the study. "So we took a radically different, reductionist approach by assembling synthetic principal components in a bottom-up fashion in a test tube." The team started with several theoretical CO -fixation routes that could result in continuous carbon cycling. But they didn't stop there. "We did not restrict our design efforts to known enzymes, but considered all reactions that seemed biochemically feasible," Erb said. Unlike DNA sequencing, where the language of life is read from the genome of an organism, DNA synthesis entails first the identification of a particular genetic element -- such as an enzyme for fixing carbon from the atmosphere -- and writing and expressing that code in a new system. In the end, they sourced, through sequencing and synthesis, 17 different enzymes from 9 different organisms across the three kingdoms of life and orchestrated these parts to achieve a proof of principle CO -fixation pathway performance that exceeds that which can be found in nature. Erb calls this the "CETCH cycle" for crotonyl-CoA/ethylmalonyl-12 CoA/hydroxybutyryl-CoA. Because it 'cetches' CO more efficiently from the atmosphere. By deploying the concept of metabolic "retrosynthesis," dismantling the reaction step by step all the way back to smaller precursors, the team juggled the thermodynamic conditions and came up with a strategy that yielded more promising results that competed favorably with natural-occurring metabolic pathways. Then they plumbed the depths of the public databases for enzymes that would support their model and selected several dozen to try out. "We first reconstituted its central CO -fixation reaction sequence stepwise, providing the ingredients to catalyze all the desired reactions. Then, by following the flux of CO we discovered which particular key reaction was rate-limiting." This turned out to be methylsuccinyl-CoA dehydrogenase (Mcd), part of a family of enzymes involved in respiration ¬- the metabolic reaction in the cells of organisms to convert nutrients like carbon into units of energy. "To overcome this limitation, we engineered the Mcd to use oxygen as an electron acceptor, to amp up the function, but this was not quite enough," said Erb. "We had to replace the original pathway design with alternative reaction sequences, used further enzyme engineering to minimize side reactions of promiscuous enzymes, and introduced proofreading enzymes to correct for the formation of dead-end metabolites," Erb said. In support of the MPI team's efforts, the DOE JGI synthesized hundreds of Enoyl-CoA Carboxylase/Reductase (ECR) enzyme variants through its Community Science Program. This enabled the MPI team to zero in on the ECR with the highest CO -fixation activity to successfully build a more efficient artificial CO fixation pathway in a test tube. "ECRs are supercharged enzymes that are capable of fixing CO at the rate of nearly 20 times faster than the most widely prevalent CO -fixing enzyme in nature, RuBisCo, which carries out the heavy lifting involved in photosynthesis," Erb said. This chemical process harnesses sunlight to turn carbon dioxide into sugars that cells can use as energy along with other natural processes on the planet and accounts for the transformation of some 350 billion tons of CO annually. Seventy years ago this phenomenon captured the imagination of early Berkeley Lab researcher Melvin Calvin who, along with Andrew Benson and James Bassham, described, in plants, algae and microorganisms, the cycle that now bears their names, and for which Calvin was awarded the Nobel Prize in 1961. This generation of researchers are concerned about how to capture excess carbon dioxide, remove it from the atmosphere and render it into energy and natural products for the economy. "Now Berkeley Lab through the DOE Joint Genome Institute, has been a major contributor to our understanding of the vast genetic diversity of microorganisms and their roles in the environment, particularly in carbon cycling," said Yasuo Yoshikuni, the head of the DNA Synthesis Science group at the DOE JGI. "By sequencing underexplored phyla from ecologically important niches, we have homed in on the genes and pathways that we now are able to synthesize in the lab to unravel novel strategies that nature uses for carbon metabolism. Identifying these genes encoding CO -fixing enzymes and their biological function, is one of the important missing pieces in the climate puzzle." Emboldened by the successful reconstitution of a synthetic enzymatic network in a test tube for the conversion of CO into organic products that is superior to chemical processes and competes with favorably with those in nature, Erb said this opens the door for other future applications. "These could include the introduction of synthetic CO -fixation cycles into organisms to bolster natural photosynthesis, or say, in combination with photovoltaics, lead the way to artificial photosynthesis, this might at the end jumpstart the design of self-sustaining, completely synthetic carbon metabolism in bacterial and algal systems." Yoshikuni looks to a future where DNA sequencing and biological functions further converge leveraging DNA synthesis. "Through DOE JGI's high-throughput sequencing capabilities coupled with the rapidly decreasing price of DNA synthesis, we continue to enable our user community in bringing to light the physiological potential of microorganisms and microbial communities. In the longer term, we hope to expect to see these test-tube results yield a new generation of real bioproducts delivered to address critical energy and environmental challenges." The broader significance of this work is to dramatically illustrate the increased role of "engineering thinking" in biotechnology, as the accelerated characterization of the biological "parts list" emerging from high throughput genome sequencing furnishes greater opportunities to reconstruct by design capacities in living organisms that address DOE mission needs in bioenergy and environment. A video from the Max-Planck Society featuring Tobias Erb discussing this project is available at http://bit.ly/ErbCETCH. This research was supported by the European Research Council, the Swiss National Science Foundation, ETH Zurich and the Max-Planck Society.


Gilbert J.A.,Argonne National Laboratory | Meyer F.,Argonne National Laboratory | Knight R.,University of Colorado at Boulder | Field D.,NERC Inc | And 3 more authors.
Standards in Genomic Sciences | Year: 2015

This report summarizes the proceedings of the Metagenomics, Metadata, Metaanalysis, Models and Metainfrastructure (M5) Roundtable at the 13th International Society for Microbial Ecology Meeting in Seattle, WA, USA August 22-27, 2010. The Genomic Standards Consortium (GSC) hosted this meeting as a community engagement exercise to describe the GSC to the microbial ecology community during this important international meeting. The roundtable included five talks given by members of the GSC, and was followed by audience participation in the form of a roundtable discussion. This report summarizes this event. Further information on the GSC and its range of activities can be found at http://www.gensc.org . © 2010 The Authors.


White O.,University of Maryland Baltimore County | Kyrpides N.,DOE Joint Genome Institute
Standards in Genomic Sciences | Year: 2015

It is widely recognized that, with the advent of very high throughput, short read, and highly parallelized sequencing technologies, the generation of new DNA sequences from microbes, plants, metagenomes is outpacing the ability to assign functions to ("annotate") all this data. To begin to try to address this, on May 18 and 19, 2010, a team of roughly fifty people met to define and scope the possibility of a first Critical Assessment of Functional Annotation Experiment (CAFAE) for bacterial genome annotation in Crystal City, Virginia. Due to the fundamental importance of genomic data to its mission, the Department of Energy (DOE) BER program hosted this workshop, funding the attendance of all invitees. The workshop was co-organized by Dan Drell and Susan Gregurick (DOE), Owen White and Nikos Kyripides. © 2010 The Authors.

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