Joint Meeting - Genomics - U.S. Department of Energy

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8 Systems Biology for DOE Energy and Environmental Missions Biofuels > Analytical and Imaging Technologies for Studying Lignocellulosic Material Degradation 7 Three-Dimensional Spatial Profiling of Lignocellulosic Materials by Coupling Light Scattering and Mass Spectrometry P.W. Bohn 1 * (pbohn@nd.edu) and J.V. Sweedler 2 (sweedler@scs.uiuc.edu) 1Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana and 2Department of Chemistry, University of Illinois, Urbana, Illinois Project Goals: The goals of the project are to: (1) develop correlated optical and mass spectrometric imaging approaches for sub-surface imaging to address the highly scattering nature of LCMs in different states of processing; (2) adapt mass spectrometric imaging protocols to enable spatially-resolved LCM characterization; (3) create surface optical and mass spectrometric measures of lignin-hemicellulosecellulose degradation at specific processing stages; and (4) correlate the in situ optical (Raman and SH-OCT) and mass spectrometric information to generate depthresolved maps of chemical information as a function of spatial position and processing time. The physical and chemical characteristics of lignocellulosic materials (LCMs) pose daunting challenges for imaging and molecular characterization: they are opaque and highly scattering; their chemical composition is a spatially variegated mixture of heteropolymers; and the nature of the matrix evolves in time during processing. Any approach to imaging these materials must (1) produce real-time molecular speciation in formation in situ; (2) extract sub-surface information during processing; and (3) follow the spatial and temporal characteristics of the molecular species in the matrix and correlate this complex profile with saccharification. To address these challenges we are implementing tightly integrated op tical and mass spectrometric imaging approaches. Employing second harmonic optical coherence tomography (SH- OCT) and Ra man microspectroscopy (RM) provides real-time in situ information regarding the temporal and spatial profiles of the processing species and the overall * Presenting author GTL chemical degradation state of the lignin heteropolymer; while MALDI and SIMS provide spatially-resolved information on the specific molecular species pro duced by pre-enzymatic processing. The goals of the approach are to: (1) develop correlated optical and mass spectrometric imaging approaches for sub-surface imaging to address the highly scattering nature of LCMs in different states of processing; (2) adapt mass spectrometric imaging protocols to enable spatially-resolved LCM characterization; (3) create surface optical and mass spectrometric measures of lignin-hemicellulose-cellulose degradation at specific processing stages; and (4) correlate the in situ optical (Raman and SH-OCT) and mass spectrometric information to generate depth-resolved maps of chemical information as a function of spatial position and processing time. 8 Identify Molecular Structural Features of Biomass Recalcitrance Using Non-Destructive Microscopy and Spectroscopy Shi-You Ding 1 * (shi_you_ding@nrel.gov), John Baker, 1 Mike Himmel, 1 Yu-San Liu, 1 Yonghua Luo, 1 Qi Xu, 1 John Yarbrough, 1 Yining Zeng, 1 Marcel G. Friedrich, 2 and X. Sunney Xie 2 GTL 1National Renewable Energy Laboratory, Golden, Colorado and 2Harvard University, Cambridge, Massachusetts Project Goals: We propose to exploit recently developed techniques to image the plant ultrastructure, map the chemistry of cell-walls, and study the time-course changes during conversion processes. These techniques are novel and developed or modified specifically for biomass characterization at the nanometer resolution. They include atomic force microscopy (AFM), solidstate NMR, small angle neutron scattering (SANS), and total internal reflection fluorescence (TIRF) microscopy. We will also use fluorescence labeling approaches to map biomass surface chemistry and to track single enzyme action in vitro and in vivo. For example, one novel technique under development is to integrate AFM with TIRF-M capability to permit us to image the cell-wall substrate at the nanometer spatial resolution and to track single cell-wall/enzyme/ cellulosome-substrate interaction. The application of these advances to the study of plant cell walls, and par-
ticularly, those focused on issues relevant to biomass conversion, is currently uncharted territory. Lignocellulosic biomass has long been recognized as a potential sustainable source of mixed sugars for fermentation to fuels and other bio-based products. However, the chemical and enzymatic conversion processes developed during the past 80 years are inefficient and expensive. The inefficiency of these processes is in part due to the lack of knowledge about the structure of biomass itself; the plant cell wall is indeed a complex nano-composite material at the molecular and nanoscales. Current processing strategies have been derived empirically, with little knowledge of the nanostructure of the feedstocks, and even less information about the molecular processes involved in biomass conversion. Substantial progress towards the cost effective conversion of biomass to fuels is contingent upon fundamental breakthroughs in our current understanding of the chemical and structural properties that have evolved in the plant cell walls which prevent its disassembly, collectively known as “biomass recalcitrance.” In nature, biomass degradation is a process of molecular interaction and reaction between plant cell wall polymers (i.e., cellulose and matrix polymers) and cellulolytic microbes and their secreted enzymes (Figure A). An integrated system (Figure B) has been set up to combine microscopic and spectroscopic modules that allow us to characterize biomass conversion processes at high spatial and chemical resolution. For example, atomic force microscopy (AFM) is used to map the surface topography of the plant cell wall and the binding of microbial cells and enzymes to the walls; total internal reflection fluorescence (TIRF) microscopy and single molecule spectroscopy is used to track the distributions and movements of labeled microbial cells and enzymes; and spectroscopy (e.g., coherent anti-stokes Raman scattering, CARS, see also Poster by Friedrich et al.) is used to monitor the resultant chemical changes in cell wall polymeric component during biochemical, as well as chemical, conversions of biomass. Preliminary results have demonstrated that integrated analysis of the same cell wall samples by diverse microscopic and spectroscopic approaches is critical for characterizing the degradation processes. In the examples illustrated, correlative imaging of AFM (Figure C-E) and TIRF-M (Figure F and G) provides molecular resolution of surface structure and chemistry of cell walls. AFM has been shown to be a powerful tool for imaging biomolecules because of its potential atomic level resolution and its ability to image surfaces under appropriately- Bioenergy buffered liquids (1). Precise measurement of cellulose microfibrils (Figure C) has been reported (1, 2) and individual cellulose chains (Figure D, Ding, unpublished) can be visualized. Using a flow-cell, the same cell wall sample can be thermo-chemically treated and imaged to monitor the structural changes that occur during pretreatment. Figure E shows particles precipitated on the wall surface after dilute acid pretreatment at 140ºC. These particles are probably lignin-carbohydrate-complexes (LCC) generated by partial hydrolysis with acid (Ding, unpublished). The cell wall can also be specifically labeled by a fluorescently-tagged carbohydrate-binding module (CBM) and imaged by TIRF-M. Figure F shows a CBM3-GFP (green fluorescence protein) labeled cell wall, in this case, the family 3 CBM specifically recognizes the planar face of cellulose (3). This image therefore reveals cellulose distribution in the cell wall. Using similar technique, individual cellulase species can be labeled with fluorescent protein and imaged using TIRF-M at the single molecule level. Figure G shows GFP-labeled Trichoderma cellobiohydrolase-I (CBH I) selectively bound on areas of an individual cellulose microfibril also labeled with CBM3-RFP (red fluorescence protein) (4). In summary, we have demonstrated non-destructive approaches to characterize the biomolecules involved in biomass conversion processes using integrated microscopy and spectroscopy. The methods are initially developed using corn stover biomass and maize plant. As a next step, we intend to employ this imaging system to characterize more energy plants, such as switchgrass and poplar wood, and their chemical and biological conversion processes to biofuels. References 1. Ding, S.-Y. & Himmel, M. E. (2006) The Maize Primary Cell Wall Microfibril: A New Model Derived from Direct Visualization. J. Agric. Food Chem. 54, 597-606. 2. Himmel, M. E., Ding, S.-Y., Johnson, D. K., Adney, W. S., Nimlos, M. R., Brady, J. W., & Foust, T. D. (2007) Biomass Recalcitrance: Engineering Plants * Presenting author 9
Page 1: Joint Meeting Genomics:GTL Awardee
Page 5 and 6: Contents Welcome ..................
Page 7 and 8: Poster Page 20 Thermophilic Electri
Page 9 and 10: Poster Page 39 Growth Rate and Prod
Page 11 and 12: Poster Page 60 MicroCOSM: Phylogene
Page 13 and 14: Poster Page 83 Experimental Mapping
Page 15 and 16: Poster Page 102 High Throughput Com
Page 17 and 18: Poster Page 120 Protein Complex Ana
Page 19 and 20: Poster Page 139 Phylogenomics-Guide
Page 21 and 22: Introduction to Workshop Abstracts
Page 23 and 24: Systems Biology for DOE Energy and
Page 25 and 26: to improve deconstruction, BESC wit
Page 27: isms for development of this CPB pr
Page 31 and 32: • Visualization of structural cha
Page 33 and 34: esolution, three-dimensional images
Page 35 and 36: 14 Single-Molecule Studies of Cellu
Page 37 and 38: while minimizing the expression of
Page 39 and 40: 7. Sokolova, T.G. et al. Thermincol
Page 41 and 42: 23 High-Throughput Screening Assay
Page 43 and 44: New Haven, Connecticut; and 3Depart
Page 45 and 46: 30 Towards Experimental Verificatio
Page 47 and 48: espect to parameters (in our case e
Page 49 and 50: photosynthetic bacteria. We will va
Page 51 and 52: Systems Environmental Microbiology
Page 53 and 54: Washington; 7 Montana State Univers
Page 55 and 56: transcriptomic data, a mutant was g
Page 57 and 58: favored Methanococcus. These result
Page 59 and 60: hydrogenotrophic methanogens for co
Page 61 and 62: at a chemical level how the obligat
Page 63 and 64: the shorter-term ESPP2 project goal
Page 65 and 66: GTL 51 Applications of GeoChip to E
Page 67 and 68: GTL 53 Comparative Metagenomics of
Page 69 and 70: To complement this research, a requ
Page 71 and 72: ties survive in uncertain environme
Page 73 and 74: use only of reliable non-transferre
Page 75 and 76: 64 Towards Genomic Encyclopedia of
Page 77 and 78: genome sequences, it is now possibl
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MR1 provided via ongoing efforts of
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strains ranges from 95.76% (S. balt
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transcriptional profiling data from
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educer Wolinella succinogenes. The
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Under O 2 -limited conditions, pyru
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dynamically generated using Java Se
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applications as well as develop a b
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tions is now underway. This computa
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simulations with randomized paramet
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experimental determination of an im
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tein. Cytochromes appear to be unde
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Therefore, it is important to under
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structures, and the functional inte
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91 Transcriptome Structure of Halob
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version with the advantages of HPF,
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(D) GmpA self-assembles into a stru
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98 New Methods for Whole-Genome Ana
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Systems Biology Research Strategy a
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communities found in coastal areas.
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challenges. For example, microbial
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oad implication for conducting prot
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variation in the AMD system, there
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scaffolds were identified by proteo
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109 Development of Mass Spectrometr
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silico from the CRISPR loci were us
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Molecular Interactions and Protein
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for high throughput characterizatio
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polyamine produced as a key interme
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Tagless purification of water solub
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characterized complexes from other
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known for homologous proteins from
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GTL 124 Protein Complex Analysis Pr
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and subjected to mass spectrometry
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of electrons to nitrogenase. Subseq
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Validation of Genome Sequence Annot
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134 Characterization of Sensor Prot
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than methods that treat functional
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Of the many functions undertaken by
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Computing Resources and Databases 1
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143 The Ribosomal Database Project
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2. It predicts which genes fill hol
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146 Genome Management Information S
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Ethical, Legal, and Societal Issues
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employment base. A key policy issue
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5. 6. McDougall, R. and Golub, A. (
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Program Websites • • • •
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A Abraham, Paul 100 Abulencia, Carl
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Fichtenholtz, Alex 20 Field, Lori 1
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Lu, Vincent 127 Lynd, L. 6 Lyons, C
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Shirodkar, Sheetal 57 Shukla, Anil
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American Type Culture Collection 93
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The complimentary use of small angl
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The reduced genomic content of M. g
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Discovery of Novel Machinery for La
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Creating a Pathway for the Biosynth
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AGENDA Sunday Evening, February 10,
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2:50-3:15 Gary Siuzdak - Scripps Re
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Paramvir Dehal, Lawrence Berkeley N
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