Progress 07/01/07 to 06/30/12
Outputs
OUTPUTS: Genetically-engineered crops are dominant components of corn-soybean production systems in the upper Midwestern USA and contribute novel plant products in crop residues to soil and water in environments where they are grown. Environmental fate of novel products of genetic engineering should consider the reactivity of these novel products at the molecular scale as well as the exposure potential and impacts at the field scale in order to address uncertainties in the ecological risk assessment for these crops. Modeling of structural changes and binding of unique proteins with reactive components representative of environmental ligands provided fundamental insight as to environmental fate of novel plant products. Protein reactivity with environmental ligands was investigated using fluorescence and X-ray diffraction as a means for understanding environmental activity of Vibrio cholera enterotoxin subunit B (CTB) as a model system under laboratory conditions. Three quinones [lawsone (2-hydroxy-1, 4- naphthoquinone), juglone (5-hydroxy-1, 4 -naphthoquinone) and AQDS (anthraquinone-2, 6- disulfonate)] were used to model reactive sites found in organic matter in order to probe changes in conformation when CTB enters agricultural environments. Fluorescence quenching studies measured binding affinity of CTB with quinones and showed weak binding as compared with a standard fulvic acid (Suwannee River 2S101F). Crystallization reactions of CTB with the model quinones confirmed that binding reactions were non-complex and were insufficient to model reactivity in the important soil retention sites such as those in fulvic acid. In order to understand how these fundamental uncertainties in novel protein environmental fate at the molecular scale translate to ecological risk assessment at the field scale, a conservative screening approach was used to evaluate the potential risk to sensitive aquatic species from corn-expressed Cry1A(b) protein occurring in a representative agricultural ecosystem from crop residues. Estimated environmental concentrations for Cry1A(b) were compared to threshold concentrations of concern for putative sensitive aquatic organisms as estimated from species sensitivity distributions. The high-end risk expressed as the combined probability of short-term exposure and acute effects to a sensitive species indicated no concern in 99% of cases with limited opportunity for chronic effects due to the rapid decline of Cry1A(b) from the environment. Sensitivity assessment for the case of Cry1A(b) aquatic risk shows that uncertainty in risk estimates for proteins derived from genetically-engineered crops can be partially resolved through improved measurement of novel protein environmental fate in soil, water, and sediment. National and international regulators and scientific experts in environmental fate and exposure characterization for genetically-engineered plants are targeted through publications and presentations. PARTICIPANTS: Jeffrey D. Wolt, Principal Investigator; Huong Tran, Graduate Research Assistant; Edward Yu, Collaborator; Robert K. D. Peterson, Collaborator TARGET AUDIENCES: National and international regulators and scientific experts in environmental fate and exposure characterization for genetically-engineered plants are targeted through publications and presentations. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Environmental fate properties of novel plant products (such as descriptors of persistence, retention, and reactivity of transgenic proteins) are important parameters for ecological risk assessment. Physical studies of protein binding and retention with components of soil organic matter help to clarify environmental fate of novel plant products; but as shown for CTB, more relevant models of environmental ligands are needed to understand reactivity of novel proteins with soil humic materials. The relevance of protein environmental fate data for ecological risk assessment was considered for the example of Cry1A(b) aquatic effects from exposure to genetically-engineered corn crop residues. While risks to aquatic species were characterized as negligible, parameters describing protein environmental fate contributed uncertainty to the risk predictions. Results of studies which resolve protein fate at both molecular and field scale show uncertainties in environmental fate parameters contributing to ecological risk assessment that will require further elaboration in terms of their measurement at the molecular scale and translation to the field scale. The outcomes of the present research provide direction as to the framework and approaches which may be used to improve environmental fate parameters important to the ecological risk assessment process for genetically-engineered crops.
Publications
- Wolt JD, Peterson RKD. 2010. Prospective formulation of environmental risk assessments: probabilistic screening for Cry1A(b) maize risk to aquatic insects. Ecotox Environ Safe 73:1182-1188.
- H Tran, J Wolt, E Yu. 2010. Characterization of the crystal of protein CTB and protein-environmental ligand complexes by using X-ray diffraction. T-331. American Crystallographic Association Annual Meeting, Chicago, IL (07/2010)
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Endoglucanases are enzymes critical for conversion of lignocellulose to ethanol for fuel production, which are rate-limiting in terms of overall cellulase activity. Corn has been transformed to express a highly stable, novel form of endoglucanase (E1) in stover as a means to improve efficiency of ethanol production. Post-harvest residues from genetically-engineered corn expressing E1 will contribute to the pool of soil endoglucanse. The degree to which E1 augments native soil cellulose activity is of importance, since the native endoglucanase compliment of soils is critical in carbon cycling and serves as an indicator of soil health. Computational biology showed that endoglucanses arise from three diverse protein families differing in folding due to arrangement of alpha-helices and beta-strands and that within a given family as little as a one amino acid change can alter stability and activity; thus the nature of novel E1 environmental fate in soils cannot be directly inferred from mesophyllic forms of endoglucnases naturally occurring in soils. The persistence and activity of residual E1 has been determined in soil microcosm studies for soils amended with bacterial, plant-solubilized, and whole plant residues of E1, and compared to native soil endoglucanse activity and to the activity of added endoglucanase from a conventional source (Aspergillus niger). A carboxymethyl cellulose substrate and dinitrosalicylic acid detection method effectively assayed endoglucanase activity in soils when optimized through use of sodium azide added during enzyme hydrolysis to increase analytical sensitivity through inhibition of glucose consumption by micro-organisms. Activity of endoglucanse added to soils rapidly declined to background levels regardless of source. Corn-expressed E1 activity persisted somewhat longer than did activity for endoglucanse from microbial sources. Whole plant E1 persisted somewhat longer than plant-solubilized E1in soil indicating an effect of the plant matrix to stabilize the enzyme. Soil endoglucanase activity does not appear to be significantly impacted from corn E1 addition at environmentally relevant concentrations. Methods and results were disseminated through publication of peer-reviewed papers, abstracts and on-line industry reports, and presentations at scientific meetings and to individual stakeholders. PARTICIPANTS: Jeffrey D. Wolt, Principal Investigator; Taner Sen, Collaborator; Ragothaman Yennamalli,Post-Doctoral Research Associate; Adam J. Kenny, Undergraduate Assistant, TARGET AUDIENCES: Scientists and regulators interested in transgenic crops especially in relation to biosafety analysis and bioprocessing, are targeted through publications and presentations. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Progress in this research makes possible a fuller understanding of how novel plant-made products may be evaluated in terms of their impact on the agricultural environment where they are grown. As a test case, using a novel form of endoglucanse expressed in corn, we apply computational biology in combination with laboratory-based measurements of enzyme activity in potentially impacted soils to understand whether the novel enzyme significantly contributes to the pool of soil enzymes which are critical to carbon cycling. The results of computational modeling of endoglucanses provides a useful template for the exploration and selection of enzymes with preferred profiles for bioprocessing and for understanding environmental fate. Methods development shows both plant and soil matrix effects on environmental persistence of endoglucanses and related cellulases can be probed with optimized assay procedures that carefully consider select assay substrate and restrict post-extraction degradation of the glucose generated as the analate. The methodology and procedures we have developed to date can be extended to the investigation of other novel plant products which may occur in agricultural soils. Regulators and product developers can use these outcomes to determine the extent to which additional data regarding environmental fate and behavior of novel plant products may be needed in considerations of product safety.
Publications
- AJ Kenny, JD Wolt. 2011. Determining persistence and ecological implications of maize-expressed transgenic endo-1,4-β-D-glucanase in agricultural soils. The Fourth International Conference, Enzymes in the Environment: Activity, Ecology and Applications. Bad Nauheim, Germany (07/2011).
- RM Yennamalli, JD Wolt, AJ Rader, TZ Sen. 2011. Sequence, structure and dynamics analysis of thermostability in endoglucanases. Biophysical Journal 100 (3, Supplement 1):536a.
- RM Yennamalli, JD Wolt, TZ Sen. 2011. Origins of thermophilicity in endoglucanases. Biophysical Journal 98 (3, Supplement 1):455a.
- Yennamalli RM, Wolt JD, Sen TZ. 2011. Dynamics of endoglucanase catalytic domains: Implications towards thermostability. J Biomol Struct Dyn 29:509-526.
- Yennamalli RM, Rader AJ, Wolt JD, Sen TZ. 2011. Thermostability in endoglucanases is fold-specific. BMC Struct Biol 11:10.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Plant expression of cellulose enzymes to enhance processing of cellulose to ethanol contributes novel, highly stable enzymes to soils in plant residues. Computational biology and physical measurements are being used to describe the environmental fate of highly thermostable endoglucanse (E1) expressed in green corn tissue. Sequence and structure similarities of thermophillic forms of endoglucanse relative to mesophilic analogs, drawn from a PDB database search, identified 118 endoglucanases categorized by three distinct folds. The fold groups are uniquely different in the arrangement of alpha-helices and beta-strands in the secondary structure. They also differ with respect to the catalytic mechanism for hydrolysis. A subset of 30 endoglucanases with sequence identity less than 70% and categorized as having either thermophilic or mesophilic origin were evaluated to identify how catalytic mechanism and overall structural fold might confer differing environmental reactivity. The amino acid compositions and intermolecular interactions for the endoglucanase fold groups show that although some amino acids are far more significant in thermophiles than mesophiles, they may not significantly alter the overall intramolecular interactions. Our results show that protein folds rather than protein families dominate in determining the specific factors responsible for protein thermostability. Elastic network models were used to identify differences in kinetically hot residues located at the substrate binding sides with cellulose and suggest that cooperative dynamics play a dominant role in thermostability of endoglucnases. Corn expressing E1 in mitochondira was characterized for analysis of environmental reactivity in comparison to a mesophylic cellulase derived from ubiquitous fungal species in soil. The dinitrosalicylic acid (DNS) method for soil cellulase activity was modified by use of carboxymethyl cellulose (CMC) substrate as a soluble, highly mineralizable carbon source. The use of CMC allowed for scaling the method to analyze for micromolar quantities of enzyme with improved specificity for endoglucanase detection at environmentally relevant concentrations, especially when sodium azide was added during CMC hydrolysis to allow glucose accumulation for improved detection. Methods and results were disseminated through publication of peer-reviewed papers, abstracts and on-line industry reports, and presentations at scientific meetings and to individual stakeholders. PARTICIPANTS: Jeffrey D. Wolt, Principal Investigator; Taner Sen, collaborator; Huong Tran, Graduate Research Assistant; Adam J. Kenny, Undergraduate Assistant. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Improved methodologies and approaches have been developed for characterizing stable proteins transgenically expressed in plants. Computational approaches have been developed that discriminate differing reactivity among endoglucanses due to amino acid sequence, fold structure, and source, thus providing a ready means to distinguish among mesostable and thermostable forms of the enzymes. An endoglucanse specific assay has been optimized for characterization of in plant expression and soil fate of highly thermostable endoglucanse. These improved approaches to characterization allow for more robust regulatory assessments and development decisions regarding fate and activity of novel plant-made proteins introduced in to agro-ecosystems.
Publications
- Wolt JD, 2009, Advancing environmental risk assessment for transgenic biofeedstock crops, Biotechnol Biofuels 2:27.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities have emphasized computational approaches to understanding fate and activity of proteins in soils and sediment, crystallization of Cholera toxin subunit B (CTB) in the presence of environmental ligands, data mining to understand the structure and activity of mesophyllic versus thermopyhillic forms of endoglucanase, and analytical method development for detection of plant-made endoglucanase in soil. These activities have been directed to post-doctoral and graduate student training and mentoring as to research techniques. NAnoscale Molecular Dynamics (NAMD) proved to be computationally unwieldy for modeling of protein conformation in simulated soil solution (a water envelope) using Escherichia coli heat labile enterotoxin subunit B (LTB) as a test case, so efforts will shift to molecular docking simulation to understand the effect of environmental ligands on protein environmental behavior. Cholera toxin subunit B (CTB) represents a well-characterized protein for modeling crystallization of protein-ligand complexes to gain insight as to protein fate and activity in soil and sediment as well as for docking simulation. Optimal conditions for crystal growth have been developed and initial crystals are being prepared for X-ray diffraction analysis. A detailed comparative analysis of thermophyllic and mesophyllic forms of endoglucanase is being explored through mining of the CAZy (Carbohydrate-Active enZymes) database to build endoglucanase protein data sets for sequence and structure analysis. The research suggests there may be fold-specific properties controlling thermophilicity in endoglucanases that will impact their environmental fate and activity. An endoglucanse assay system based and glucose release from carboxymethyl cellulose has been validated and is being adopted for studies of thermostable E1 endoglucanse fate and activity in soil. Methods and results were disseminated through publication of peer-reviewed papers, abstracts and on-line industry reports, and presentations at scientific meetings and to individual stakeholders. PARTICIPANTS: Project collaborators: Taner Sen, Michael Thompson, Ali Tabatabai, Edward Yu. Graduate student trainees: James Delgado, Huong Tran. Post-doctoral trainee: Ragothaman Yennamalli. Undergraduate trainee: Adam J. Kenny. Partner organization: USDA-ARS, Corn Insects and Crop Genetics Research, Crop Genome Informatics Laboratory. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Computational simulation, data mining, and crystallization reactions together represent a unique fundamental approach to probing the environmental behavior of novel proteins in environmental matrices. The knowledge gained will lead to better first tier approximations of protein degradation for the environmental risk assessment for transgenic plant products. The interface of this research with more conventional environmental fate studies in graduate training and research will lead to a more robust set of approaches for understanding the nature and behavior of unique biological molecules introduced into agricultural environments.
Publications
- Wolt JD. 2009. Advancing environmental risk assessment for transgenic biofeedstck crops. Biotechnology for Biofuels 2:27.
- Shelton AM, SE Naranjo, J Romeis, RL Hellmich, JD Wolt, BA Federici, R Albajes, F Bigler, EPJ. Burgess, GP Dively, AMR Gatehouse, LA Malone, R Roush, M Sears, F Sehnal, Na Ferry, HA Bell. 2009. Appropriate analytical methods are necessary to assess nontarget effects of insecticidal proteins in GM crops through meta-analysis. Environ. Entomol. 38(6):1533-1538.
- Shelton AM, SE Naranjo, J Romeis, RL Hellmich, JD Wolt, BA Federici, R Albajes, F Bigler, EPJ Burgess, GP Dively, AMR Gatehouse, LA Malone, R Roush, M Sears, and F Sehnal. 2009. Setting the record straight: a rebuttal to an erroneous analysis on transgenic insecticidal crops and natural enemies. Transgenic Research 18:317.322.
- Wolt JD, P Kees, A Raybould, JW Fitzpatrick, M Burachik, A Gray, SS Olin, J Schiemann, M Sears, and F Wu. 2009. Problem formulation in the environmental risk assessment for genetically modified plants. Transgenic Research DOI 10.1007/s11248-009-9321-9.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Activities have emphasized design and conduct of experiments and modeling to describe fate of transgenic products in soils and sediments. The principle focus of activity was directed to graduate student training and mentoring as to research techniques. Computational modeling of protein conformation in simulated soil solution (a water envelope) was initiated for Escherichia coli heat labile endotoxin subunit B (LTB) and for the closely homologous Cholera toxin subunit B (CTB) using NAnoscale Molecular Dynamics (NAMD). Limitations in simulation of ambient temperature systems were identified and are being addressed in order to identify regions of the proteins which are labile to conformational changes upon interaction with soil colloids, and which will alter activity and/or degradability of these proteins. Initial studies of persistence and degradation of bacterial and maize-expressed LTB and CTB in soil and water have been completed and companion studies are being designed to further investigate dynamics of sorption-degradation relationships. A key product arising from this project has been the development of a collaborative network of researchers on the Iowa State campus who have interest in the environmental molecular science of transgenic plant product environmental fate and effects. The collaborative links amongst this group of soil scientists, molecular and computational biologists, and environmental toxicologists are intended to provide improved graduate training and research funding opportunities that advance research approaches for understanding and describing the ultimate fate and activity of unique plant-made proteins intended for use as industrial enzymes and pharmaceutical actives. An environmental fate laboratory is being established and staffed to serve as a focal point for graduate training and research in environmental molecular science. PARTICIPANTS: Project collaborators: Kan Wang, Joel Coats, Taner Sen, Michael Thompson, Ali Tabatabai. Graduate student trainees: James Delgado, Huong Tran. Post-doctoral trainee: Sule Karaman. Partner organization: USDA-ARS, Corn Insects and Crop Genetics Research, Crop Genome Informatics Laboratory. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Experiments comparing soil dissipation of LTB and CTB in both bacterial and plant-made forms have provided firm evidence of the effect of plant matrix (corn kernel endosperm starch) in facilitating a delay in the dissipation of protein. This knowledge will lead to better understanding of how to design, conduct, and interpret protein degradation data as one facet of the environmental risk assessment for transgenic plant products. Networking of activities amongst researchers with diverse backgrounds is changing the conditions whereby graduate training and research will be conducted to better address questions regarding the likelihood of exposure and consequences of introducing novel plant-made products into the ecosystem.
Publications
- Kosaki H, Coats, J., Wang, K., and Wolt, J. 2008. Persistence and degradation of maize-expressed vaccine protein, Escherichia coli heat-labile enterotoxin subunit B, in soil and water, Environmental Toxicology and Chemistry 27:1227-1236.
- Wolt, J.D., Sen, T., Tabatabai, M.A., and Tran, H. 2008. Experimental approaches for determining environmental fate of transgenic plant proteins. Entomological Society of America Annual Meeting, Reno NV.
- Wolt, J.D. 2008. Framework for environmental risk assessment of GM crops: Problem formulation as an essential first step. Convention on Biodiversity, COP 9 MOP 4, Bonn, Germany.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Using a consensus-building we are engaged with scientists and regulators under sponsorship of the International Life Sciences Institute and the International Organization for Biocontrol West Palaearctic Regional Section in efforts to elaborate the process of environmental risk assessment (ERA) of transgenic crops. Specific emphasis is being given to problem formulation, the tiered analysis process applied to GMAPs, and ecotoxicity testing strategies for non-target arthropods. Laboratory activities have centered on method development for detection of both wild-type and transgenic enzymes in soil and water matrices as proof of concept for investigation of how corn transgenic enzymes intended for biofuel production may impact soil carbon sequestration. A problem formulation and initial exposure assessment describing environmental loads of thermostable alpha-amylase from transgenic high-amylase corn was conducted. Consensus activities centered on the process of environmental
risk assessment for transgenic crops we have helped to increase shared knowledge amongst opinion leaders in the global regulatory process for genetically modified organisms. Actions taken have included (1) a consensus white paper on the ERA process for non-target organisms which is now in press and (2) technical leadership of a two-day expert consultation on problem formulation which has resulted in a presentation at the Society for Risk Analysis and the drafting of a white paper for publication. A presentation at the 2nd Annual Symposium of the Agricultural Risk Assessment (AGRA) described for a broad spectrum of scientists, regulators, and federal research managers the rationale and approach we are undertaking in the development of a tiered process for evaluation of GMAP fate in soil.
PARTICIPANTS: Dr. Sule Karaman, Post-doctoral Research Associate, Biosafety Institute for Genetically-Modified Organisms, Iowa State University. James Delgado, PhD student, Interdepartmental Toxicology Program, Iowa State University. Hirofumi Kosaki, MS student, Interdepartmental Toxicology Program, Iowa State University.
TARGET AUDIENCES: Federal and international regulatory authorities tasked with responsibility for environmental risk assessment of genetically-modified crops. Scientists conducting research in support of the environmental risk assessment of genetically-modified crops. Interested and affected parties to regulatory determinations regarding environmental releases of genetically modified crops.
Impacts
There is an elevated degree of understanding amongst opinion leaders in the area of plant biotechnology risk assessment research and regulation of the impact of data generation strategies and data synthesis on policies for evaluating exposure and risk associated with the environmental fate and effects of transgenic plant deployments within agro ecosystems.
Publications
- Wolt, J.D. and Karaman, S. 2007. Estimated environmental loads of alpha-amylase from transgenic high-amylase maize. Biomass and Bioenergy 31:831-835.
- Chapotin S.M. and Wolt, J.D. 2007. Genetically engineered plants for the bioeconomy. Transgenic Research 16:675-688.
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