Principal Investigator: Wellington Muchero
AbstractUsing willow, a widely used biofuel feedstock and pioneer species with increasing presence in the warming Arctic region, we will identify and characterize unique host-derived genetic factors that allow select microbes to successfully evade plants defense mechanisms. The research presents a unique opportunity to couple new growth-promoting microbes with willow to increase carbon sequestration in the vulnerable Arctic and improve plant biomass yields for the sustainable production of cellulosic biofuels.
The productivity of bioenergy crop Sorghum grown in marginal lands can be significantly improved by biological nitrogen fixation (BNF) using endophytic diazotrophs. However, it has been a challenge to establish stable colonization by diazotrophs in the field. To address this challenge, we propose a multi-generation microbiome selection approach to generate BNF consortia that can effectively colonize plant endospheres. Sorghum plants will be grown in nitrogen-poor soils and their incorporation of 15N2 fixed by root endophytes will be monitored during the selection process. Plant microbiomes will be collected and used to inoculate Sorghum plants in the next round of selection for a total 5 rounds. By reducing soil nitrogen availability in each round, we expect to gradually improve the BNF performance of plant microbiomes. Integrated -omics and imaging will be used to characterize the BNF consortia and the plant hosts to test a bacterial association hypothesis and a host selection hypothesis. The results are expected to define the symbiotic associates of diazotrophs with Sorghum host, and reveal molecular changes of the host plant during colonization and nitrogen fixation by the BNF microbiome. This study will generate BNF consortia for Sorghum and provide fundamental understanding of host-microbiome interaction under nitrogen limitation.
Fungi are one of the most important groups of organisms on the planet playing vital roles in the biosphere. The development of effective methods for genetic engineering of fungi over the past two decades has demonstrated the importance of such tools to alter the detrimental and beneficial activities and metabolic processes of fungi. Nonetheless, detailed knowledge into the molecular biology and biochemistry is only available for a few fungal models mostly representative of the group of the Ascomycetes. Full genome sequencing efforts by the DOE Joint Genome Institute (e.g., 1KFG, MycoCosm projects) are rapidly increasing to better explore the entire fungal kingdom. The sparsity of Basidiomycete and Zygomycete biochemical knowledge can be partly explained by a lack of well-developed genetic tools. Basidiomycetes and Zygomycetes, the two major phyla besides Ascomycetes, are important in medicine, industry, agriculture and environmental research. Therefore, we propose to fill this gap and to establish an easy and versatile fungal genetic engineering platform that works for all major phyla (i.e., Ascomycetes, Basidiomycetes and Zygomycetes). The known CRISPR-Cas9 system has been successfully adapted to a wide range of organisms. Recently, the system was successfully implemented in yeasts. By creating a simple and versatile fungal CRISPR-Cas9, this project will provide an efficient high-throughput system and methodology for directed mutagenesis into single or multiple loci applicable to diverse marker-free fungi. Such a capability will position ORNL in the forefront of the fields of synthetic and systems biology.
Terpenes have the highest energy density and affordable conversion efficiency of any biofeedstock. Research proposed here focuses on maximizing production, reducing volatilization and optimizing terpene content in Eucalyptus leaf oil glands (OG) for the creation of advanced fungible biofuel production in the U.S. By combining metabolic characterization, neutron scattering and transcript profiling, we will gain molecular-level insights into the determinant properties for synthesis and storage of terpenes in oil glands. Collectively, defining the genetic mechanisms of Eucalyptus leaf OG structure and chemistry will enable future applications of these resources for the genetic improvement of foliar OG number and volume, coupled with enhanced synthesis and storage of volatile and non-volatile terpenes. The scientific objectives are to identify individual Eucalyptus genotypes that have high potential for terpene production and to determine the genes that control biochemical and anatomical features of favorable terpene production.
Director: Paul Gilna, ORNL
FA1 Lead: Debra Mohnen, University of Georgia
ORNL FA1 Co-investigators: Jay Chen, Udaya Kalluri, Wellington Muchero, Priya Ranjan, Tim Tschaplinski, Gerald Tuskan , Xiaohan Yang
The BioEnergy Science Center (BESC) is a multi-institutional (18 partner), multidisciplinary research (biological, chemical, physical and computational sciences, mathematics and engineering) organization focused on the fundamental understanding and elimination of biomass recalcitrance. BESC is one of three Bioenergy Research Centers established by DOE's Office of Science in 2007 to accelerate research toward the development of cost-effective advanced biofuels.
BESC's approach to improve accessibility to the sugars within biomass involves 1) designing plant cell walls for rapid deconstruction and 2) developing multitalented microbes for converting plant biomass into biofuels in a single step (consolidated bioprocessing). Addressing the roadblock of biomass recalcitrance will require a multiscale understanding of plant cell walls from biosynthesis to deconstruction pathways. This integrated understanding would generate models, theories and finally processes that will be used to understand and overcome biomass recalcitrance.Project website: www.bioenergycenter.org
Engineering CAM Photosynthetic Machinery into Bioenergy Crops for Biofuels Production in Marginal Environments
Principal Investigator: John C. Cushman, University of Nevada, Reno
Co-investigators: Anne Borland, Jay Chen, James Hartwell, Madhavi Martin, Karen Schlauch, Tim Tschaplinski, Gerald Tuskan, David Weston, Xiaohan Yang
Crassulacean acid metabolism (CAM) is a photosynthetic CO2 fixation pathway that maximizes water use efficiency (WUE) many times relative to C3 species by using a temporal CO2 pump. Thus, CAM provides an excellent opportunity to engineer both enhanced photosynthetic performance and WUE into bioenergy crops. The proposed research will provide a comprehensive understanding of the enzymatic and regulatory pathways required to engineer CAM photosynthetic machinery into Populus. The methods employed will include deep transcriptome sequencing (RNA-Seq) and high-throughput metabolic profiling of leaf mesophyll cells and stomatal guard cells to identify regulators of the nocturnal opening and daytime closure of stomata that underpins the high WUE of CAM plants in order to create co-expression models. Additional deep genome sequencing combined with chromatin-immunoprecipitation (ChIP-Seq) experiments will be conducted to characterize the transcriptional regulatory networks needed for the circadian clock regulation of CAM. Once characterized, metabolic pathway components of 'carboxylation' and 'decarboxylation' modules will be assembled using an iterative cloning system, and CAM modules will be assembled singly and in combination into a predefined single locus of the target plant genome. Modules will be expressed under the control of circadian clock controlled, drought-inducible promoters in both the readily transformable model Arabidopsis and the important bioenergy crop Populus to promote maximal productivity. Resulting plants will be tested under both control and drought stress conditions for transgene expression, biochemical signatures of CAM, CO2 assimilation, stomatal conductance and transpiration rates, leaf carbon balance, level/mode of CAM activity, biomass productivity and quality, and integrated WUE.Project website: cambiodesign.org
Our inability to accurately represent plant functional traits (e.g., those traits governing photosynthesis) for a wide array of taxa and the interaction of those traits with variable environmental conditions are considered key uncertainties in land-surface models including the DOE BER funded Community Land Model (CLM). Given the importance of this issue, it is unfortunate that the scientific community is not currently leveraging advances in genomics and genetics to better predict plant traits that govern species performance under various climatic conditions. Here, we will design a laboratory system and an instrumented field plot to: 1) develop advanced genomic modeling approaches to predict trait distributions for species critical to C cycling, 2) deploy this model as an open source service within the KBase knowledgebase project that is interfaced with climate models and, 3) test the output of model runs with laboratory and field based manipulations within a critical ecosystem. The proposed project will eliminate the current disconnection between BER genomics (BSSD) and Climate (CESD) based research, and thereby set the precedent where advances from biological system research are brought to bear in climate system research.
Principal Investigator: Mitch Doktycz
ORNL Co-investigators (Plant): Jay Chen, Udaya Kalluri, Jessy Labbé, Wellington Muchero, Priya Ranjan, Tim Tschaplinski, Gerald Tuskan, David Weston, Xiaohan Yang
The Plant-Microbe Interfaces (PMI) project is a Scientific Focus Area directed towards understanding the dynamic interface that exists between plants, microbes and their environment. A specific focus is on defining the genetic bases of molecular communication between Populus and its microbial consortia. Understanding the inherent chemical and physical processes involved will facilitate natural routes to the cycling and sequestration of carbon in terrestrial environments, ecosystem response to climate change, and the development and management of renewable energy sources.
The project integrates expertise in the areas of plant genomics, fungal and bacterial research, fungal ecology, analytical tool development and computational biology and is based at the Oak Ridge National Laboratory, with collaborators at the University of Washington, Duke University, and INRA - Nancy (France). The project is a Foundational Genomics Scientific Focus Area supported by the Genomic Science Program of the Office of Biological and Environmental Research of the U.S. Department of Energy.
Project website: pmi.ornl.gov