Source: NORTH CAROLINA STATE UNIV submitted to NRP
BIOTECHNOLOGICAL APPLICATIONS OF EXTREMOPHILES FOR BIOFUEL PRODUCTION, BIOREMEDIATION, AND CROP IMPROVEMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1001331
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2013
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Microbiology
Non Technical Summary
This project is focused on using extremophiles for biotechnical applications for improved biofuel production, bioremediation and crop improvement. Extremophiles are microorganisms that are capable of thriving in diverse extreme environmental conditions (i.e. high or low temperatures, high salinity, acidic or alkaline environments) and as a result these microbes have genes/proteins which can have attributes that are valuable to the development of novel biotechnological applications. Currently there are biologically imposed limitations to plant metabolism that constrain plant productivity, and thereby the amount of biofuel that can be generated from these plants. Several of the goals of this research (Objectives 1, 2, and 5) are targeted at improving plant productivity using select extremophile genes. Specifically, we are addressing the limitations of plant-based carbon fixation by designing a new carbon fixation cycle that can be used in plants in conjunction with the traditional Calvin-Benson cycle to convert atmospheric carbon dioxide to plant biomass and biofuel precursor molecules (Objective 1). In addition, extremophile genes are being used to enhance carbon dioxide uptake and nutrient recycle in algal biofuel systems (Objective 2). We are also introducing an antioxidant gene coding for superoxide reductase (SOR) into plants to improve the ability of plants to cope with environmental stresses such as temperature, drought, metal, radiation and pathogen stresses (Objective 5). Biofuels can also be generated from conversion of waste gas streams (CO and CO2 containing synthesis gas that can be produced from gasification of waste agriculture biomass) using microbial catalysts. We have developed a synthesis gas fermentation platform employing a modified strain of clostridium ljungdahlii and are using gene expression data and fermentation profiles to further improve this system (Objective 3). Much progress has also been made in recent years to use microbial catalysis to aid in critical efforts of biodecontamination of toxic substances in the environment. In particular recent research has demonstrated the great potential for using enzymes from extremophiles to provide efficient biocatalysis under a variety of potentially harsh process conditions. Continued research is required to identify and characterize suitable extremophile enzymes in order to develop robust microbial catalysis processes that would otherwise be impossible using standard enzyme systems or chemical methods. In particular, we are investigating the use of a proline dipeptidase enzyme called prolidase from the high temperature organism Pyrococcus furiosus to degrade toxic organophosphate compounds found in some nerve agents such as soman and sarin and in some pesticides (Objective 4). We have bioengineered highly active recombinant versions of the Pyrococcus prolidase enzymes that are active over a broad range of temperatures and are evaluating their ability to degrade nerve agent and pesticide analogs.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20322991100100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
2299 - Miscellaneous and new crops, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
There are five major objectives associated with this project, which are as follows: 1) Generate a novel synthetic carbon fixation cycle composed of select microbial enzymes for use in plant systems to enhance plant biomass and fuel precursor production. 2) Use of extremophile genes to optimize fatty acid production in microalgae for biofuel production 3) Use of Immobilized Clostridium ljungdahlii in Engineered Biofilms for Synthesis Gas Fermentation to Liquid Fuels 4) Use enzyme structure information to rationally bioengineer thermostable Pyrococcus prolidases for use in organophosphate nerve agent and pesticide detoxification. 5) Engineer plants (Arabidopsis thaliana, Camelina sativa) with an extremophile antioxidant enzyme, superoxide reductase (SOR), to generate pants with improved response to environmental stresses such as temperature, drought, metal, radiation and pathogen stresses.
Project Methods
For Objective 1 to generate a novel synthetic carbon fixation cycle composed of select microbial enzymes to enhance plant biomass and fuel precursor production, we are using standard molecular biology techniques to clone, over-express and purify candidate cycle enzymes. These enzymes are being evaluated for function using a combination of spectrophotometric, NMR and LC-MS based methods. We are using modified Gateway vectors to introduce the synthetic carbon fixation cycle enzymes into plant genomes (tobacco, camelina) and will be using Western analysis and activity assays to demonstrate in planta expression and function of the cycle enzymes. Ultimately we will be using LiCOR analysis, growth metrics and oil production analysis to evaluate for increased carbon fixation capabilities, biomass production and oil production of the transgenic plants. For Objective 2 to develop a scalable algal-based photosynthetic biorefinery using genetically improved algae for production of algal oils for biofuel conversion, the following tasks/methods will be employed: • Bioengineering Dunaliella viridis with bacterial high-affinity CO2/bicarbonate transporters and carbonic anhydrases to enhance microalgal CO2 uptake as well as modification of algae productivity using recycled biomass for N-, P-, and S- supplementation • Development and validation of a kinetic model describing microalgal growth and lipid production in response to CO2, light, pH, recycled nutrients and temperature • Design of a small-scale (10 L) PSBR to inform the development a scalable dynamic reactor model using computational fluids dynamic simulation. Micron-scale chemical sensor beads will be developed to evaluate the spatial distribution of light in the operating PSBR • Evaluation of experimental harvesting (e.g., microfiltration) and lipid recovery methods (osmotic lysis) and novel N and P recycle methods for reutilization of algal biomass • Development of an analytical framework for life-cycle analysis (LCA) of our bio fuel production system by including flexible and scalable cost and life-cycle inventory process models that will generate a robust decision tool for optimizing reactor design and operation For Objective 3 to understand the metabolic regulation occurring in the autotrophic acetogen Clostridium ljungdahlii for improving clostridial biofuel production from waste gases, we are using DNA sequencing, RNA sequencing, quantitative PCR, and fermentation analysis methods. As part of this project, we have developed a modified strain of wild type C. ljungdahlii termed C. ljungdahlii OTA1 that has superior waste gas utilization and ethanol production capabilities. We are using whole genome sequencing to identify discreet changes in the OTA1 strain genome and RNA sequencing to establish differences in gene expression under different growth conditions. Spectrophotometry, GC-MS and HPLC analysis are used to evaluate Clostridium fermentation performance. For Objective 4 to use enzyme structure information to rationally bioengineer thermostable Pyrococcus prolidases for use in organophosphate nerve agent and pesticide detoxification, we are employing random and targeted mutagenesis strategies, a selective screening technique for isolation of mutant prolidases with desired characteristics, and spectrophotometric assays to evaluate activity. For Objective 5 to engineer plants (Arabidopsis thaliana, Camelina sativa) with an extremophile antioxidant enzyme, superoxide reductase (SOR) to provide stress resistance, we are using a variety of plant stress test methods and activity assays to evaluate fitness of the transgenic plants.

Progress 10/01/13 to 09/30/18

Outputs
Target Audience:Our research efforts principally benefit the Department of Energy, the National Science Foundation the Department of Agriculture and the Department of Defense, as our research provides important information about development of an effective auxiliary carbon fixation pathway for use in biofuel crop plants, development of algal strains with improved oil and co-product production and biodecontamination systems for detoxification of organophosphorus-based chemical weapons. Our research efforts have also served as a training platform for providing mentored research experience to postdoctoral scholars, as well as graduate, undergraduate, and high school students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported training of one postdoctoral scholar, three graduate students, three undergraduates and 4 high school students. How have the results been disseminated to communities of interest?Our project results have been disseminated through public lectures, research symposia presentations, and published research articles. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major Project Goal Accomplishments: 1) Generate a novel synthetic carbon fixation cycle composed of select microbial enzymes for use in plant systems to enhance plant biomass and fuel precursor production. Camelina is an excellent biofuel crop system because it grows on poor soils with little water and a short growth period and produces oil-containing seeds with an oil-blend amenable to conversion to transportation fuels, including jet fuels. However,Camelina is a C3 type plant, limiting its carbon fixation capabilities. Objective 1 of this project will generate a new synthetic pathway to increase CO2 assimilation in the chloroplast (condensed, reverse TCA cycle, crTCA cycle) through expression of a suite of genes from plant, bacterial, and archaeal origin. The pathway will be integrated with other transgenes to increase the CO2 concentration inside the chloroplast (CO2 - transporter AQP1), reduce the photorespiratory energy loss (bacterial bypass), and increase photosynthetic efficiency by reducing reactive oxygen species (superoxide reductase). In this reporting period we were able to 1) demonstrate the in vitro function of the full cycle using LC-MS and activity assays, 2) generate two constructs, one which contains the first two enzymes of the cycle and the second which expresses the last three enzymes of the cycle for transformation of Camelina sativa, 3) use transient tobacco transformation to demonstrate in planta activity of four of the five crTCA cycle enzymes. 2) Use of extremophile genes to optimize fatty acid production in microalgae for biofuel production. Algal oils are an ideal feedstock for biofuels production, offering very high production density and the ability to use marginal water (municipal waste, brackish water, etc.) and reuse CO2 emitted from coal-fired power plants. However, there are a number of technical challenges associated with efficiently exploiting algae's inherent advantages as a feedstock. One such challenge is that lipid synthesis in microalgae is a highly regulated process than can limit the accumulation of algal lipids. Fatty acid synthesis (FAS) is a highly conserved process in microalgae, archaea and bacteria, in which the pathway that functions in chloroplastic FAS in microalgae also functions similarly in bacteria and archaea. The first committed step in microalgal FAS is the conversion of acetyl CoA to malonyl CoA by acetyl CoA carboxylase (ACCase). This step is also the primary regulated and rate-limiting step for FAS in microalgae, so an increase in ACCase activity should result in increased FAS. Indeed, increased levels of ACCase expression in bacterial systems lead to elevated rates of FA production. Therefore, as a means to overcome transcriptional repression and alleviate feedback inhibition of microalgal ACCase, we propose to express select archaeal and bacterial ACCase genes in the target Dunaliella strain to maximize its FAS. In addition, expression of thioesterases relieves acyl-ACP inhibition of ACCase and allowed increased FAS even under otherwise FASinhibiting conditions. We will also express a type I thioesterase from C. salexigens (Csal_2620) in Dunaliella in an effort to further limit feedback inhibition of its ACCase. In this reporting period, we were able to 1) demonstrate recombinant expression and activity of the halophilic bacterial ACCase from Chromohalobacter salexigens that will be expressed in Dunaliella, 2) demonstrate recombinant expression and biochemically characterize C. salexigens TesA and show that it can effectively convert acyl-coAs to free fatty acids and 3) generate a C. salexigens TesA expression construct with which to transform Dunaliella. 3) Use of Immobilized Clostridium ljungdahlii in Engineered Biofilms for Synthesis Gas Fermentation to Liquid Fuels. Our model synthesis gas fermentation system is a novel combination of the well-established biofiltration process and the immobilization methods developed by our research group. The proposed activities will use C. ljungdahlii synthesis gas fermentation to define the physiological state of the immobilized cells in our gas phase reactor, characterizing reactivity and response to the most significant trace inhibitors in synthesis gas. In this reporting period we were able to 1) generate and analyze the genome sequence of our wildtype and an improved lab derived strain (OTA1) of Clostridium ljungdahlii, 2) use the genome sequence analysis to identify the mutations that exist in the OTA1 genome which may confer its improved ethanol production in comparison to wildtype C. ljungdahlii, 3) conduct growth, fermentation, enzyme assay and RNA seq analysis of wildtype C. ljungdahlii exposed to low oxygen (0-8%) to evaluate the metabolic response of C. ljungdahlii to oxygen and to determine why oxygen exposure enhances ethanol production by C. ljungdahlii. 4) Application of Extremophile Enzymes for Organophosphorus Compound Detoxification. In the past year we have successfully expressed P. horikoshii prolidase in the microalgae Nannochloropsis oceanica and are currently biochemically characterizing the algal expressed prolidase. 5) Use of reactive oxygen detoxification enzymes from the hyperthermophilic archaeon Pyrococcus furiosus to generate transgenic plants with increased tolerance to harsh environmental conditions. In this reporting period we have introduced the P. furiosus antioxidant enzyme into cyanobacteria and dogwood and have evaluated its efficacy in providing protection against environmental stress. Both transgenic cyanobacteria and dogwood appear to benefit from SOR expression.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Smith-Moore CM, Grunden AM. 2018. Bacteria and Archaea as a source of traits for enhanced plant phenotypes. Biotechnology Advances. 36(7):1900-1916.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kitchener, RL, Grunden AM. 2018. Methods for enhancing cyanobacterial stress tolerance to enable improved production of biofuels and industrially relevant chemicals Applied Microbiology and Biotechnology. Appl. Microbiol. Biotechnol. 102(4):1617-1628. doi: 10.1007/s00253-018-8755-5.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: . Somalinga V, Klemmer H, Arun A, Wapshott H, Mathews S, Grunden AM. 2018. Cloning, Over-Expression, and Purification of Carbonic Anhydrase from an Extremophilic Bacterium: An Introduction to Advanced Molecular Biology. The American Biology Teacher, 80:29-34.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Duckworth OW, Andrews MY, Cubeta MA, Grunden AM, Ojiambo PS. 2017. Revisiting Graduate Student Training to Address Agricultural and Environmental Societal Challenge. Agric. Environ. Lett. 2:170019, doi:10.2134/ael2017.06.0019.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Karam AL, McMillan CC, Lai YC, de Los Reyes FL, Sederoff HW, Grunden AM, Ranjithan RS, Levis JW, Ducoste JJ. 2017. Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests. J Vis Exp. 14;(124). doi: 10.3791/55545
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Whitham JM, Schulte MJ, Bobay BG, Bruno-Barcena JM, Chinn MS, Flickinger MC, Pawlak JJ, Grunden AM. 2017. Characterization of Clostridium ljungdahlii OTA1: a non-autotrophic hyper ethanol-producing strain. Appl. Microbiol. Biotechnol. 101(4):1615-1630. DOI:10.1007/s00253-016-7978-6
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ortiz MJF, Grunden AM, Hyman M, Call D. 2018.Performance of Nitrogen-Fixing Anodic Biofilms for Potential Ammonia Production in Microbial Electrochemical Technologies. 2018 National ACS Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Cuebas-Irizarry M, Gray R, Grunden AM. 2018. Evaluating Lignin Degradation Potential of Bacterial Isolates from Carpenter Bees. 2018 North Carolina Branch American Society for Microbiology Symposium.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wapshott H, Vazquez G, Grunden AM. 2018. Developing OleTJE-Superoxide Reductase enzyme fusions to facilitate efficient production of terminal alkenes for use as renewable drop-in transportation fuels. 2018 NIH MBTP Symposium.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Greenstein J, Wapshott H, Hamilton P, Grunden AM. 2017. Two-step enzymatic conversion of algal triacylglycerides to hydrocarbons. Frontiers in Biorefining Conference. St Simon's Island, Georgia.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grunden AM, Biology Program Seminar Series, University of North Carolina, Chapel Hill, NC
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grunden AM, Microbiology Seminar Series, University of Georgia, Athens, GA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grunden AM, VIB-NC State Strategic Alliance Workshop, Ghent Belgium
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grunden AM, Integrative Molecular Plant Systems NSF REU program, NCSU, Raleigh NC
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grunden AM, USDA BeeMore REE program, NCSU, Raleigh, NC


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Our research efforts principally benefit the Department of Energy, the National Science Foundation the Department of Agriculture and the Department of Defense, as our research provides important information about development of an effective auxiliary carbon fixation pathway for use in biofuel crop plants, development of algal strains with improved oil and co-product production and biodecontamination systems for detoxification of organophosphorus-based chemical weapons. Our research efforts have also served as a training platform for providing mentored research experience to postdoctoral scholars, as well as graduate, undergraduate, and high school students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported training of two postdoctoral scholars, three graduate students, four undergraduates and four high school students. How have the results been disseminated to communities of interest?Our project results have been disseminated through public lectures, research symposia presentations, and published research articles. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will undertake the following activities as indicated for each of our major project goals: Goal 1: we will 1) finish the in vitro characterization of our crTCA cycle and will submit our findings for publication, 2) will characterize camelina sativa lines that have been transformed with both of the crTCA cycle expression constructs, and 3) will evaluate the effect of the crTCA cycle expression on plant biomass and oilseed production. Goal 2: we will 1) transform the microalgae Dunaliella or other sutiable microalgae with the TesA thioesterase from the halophilic bacterium C. salexigens to improve fatty acid synthesis in the transgenic algae, 2) will transform Dunaliella with industrial extremozymes to provide high value co-product production in the algae, and 3) will identify and characterize enzymes that can be used for nutrient recycle for in a photobioreactor system. Goal 3 we will 1) determine which of the identified mutants in the improved OTA1 C. ljungdahlii strain are responsible for its enhanced ethanol production. Goal 4: we will characterize the extremophile OP degradation enzymes we have expressed in our algae production system and compare the the activity of the algae expressed enzymes to bacterial expressed versions. Goal 5:we will further characterize the SOR expressing cyanobacteria and dogwood and report our findings in journal articles.

Impacts
What was accomplished under these goals? Project Justification/Impact: This project is focused on using extremophiles for biotechnical applications for improved biofuel production, bioremediation and crop improvement. Extremophiles are microorganisms that are capable of thriving in diverse extreme environmental conditions and as a result these microbes have genes/proteins which can have attributes that are valuable to the development of novel biotechnological applications. Currently there are biologically imposed limitations to plant metabolism that constrain plant productivity, and thereby the amount of biofuel that can be generated from these plants. Several of the goals of this research (Obj. 1, 2, and 5) are targeted at improving plant productivity using select extremophile genes. Specifically, we are addressing the limitations of plant-based carbon fixation by designing a new carbon fixation cycle that can be used in plants in conjunction with the traditional Calvin-Benson cycle to convert atmospheric carbon dioxide to plant biomass and biofuel precursor molecules (Objective 1). In addition, extremophile genes are being used to enhance carbon dioxide uptake and nutrient recycle in algal biofuel systems (Obj. 2) and to decontaminate organophosphate based pesticides and chemical warfare agents (Obj. 4. We are also introducing an antioxidant gene coding for superoxide reductase (SOR) into plants to improve the ability of plants to cope with environmental stresses such as temperature, drought, metal, radiation and pathogen stresses (Obj. 5). Biofuels can also be generated from conversion of waste gas streams (CO and CO containing synthesis gas that can be produced from gasification of waste agriculture biomass) using microbial catalysts. We have developed a synthesis gas fermentation platform employing a modified strain of Clostridium ljungdahlii and are using gene expression data and fermentation profiles to further improve this system (Obj. 3). Major Project Goals: 1) Generate a novel synthetic carbon fixation cycle composed of select microbial enzymes for use in plant systems to enhance plant biomass and fuel precursor production. Camelina is an excellent biofuel crop system because it grows on poor soils with little water and a short growth period and produces oil-containing seeds with an oil-blend amenable to conversion to transportation fuels, including jet fuels. However, Camelina is a C3 type plant, limiting its carbon fixation capabilities. Objective 1 of this project will generate a new synthetic pathway to increase CO2 assimilation in the chloroplast (condensed, reverse TCA cycle, crTCA cycle) through expression of a suite of genes from plant, bacterial, and archaeal origin. The pathway will be integrated with other transgenes to increase the CO2 concentration inside the chloroplast (CO2-transporter AQP1), reduce the photorespiratory energy loss (bacterial bypass), and increase photosynthetic efficiency by reducing reactive oxygen species (superoxide reductase). In this reporting period we were able to 1) demonstrate the in vitro function of the full cycle using LC-MS and activity assays, 2) generate two constructs, one which contains the first two enzymes of the cycle and the second which expresses the last three enzymes of the cycle for transformation of Camelina sativa, 3) use transient tobacco transformation to demonstrate in planta activity of four of the five crTCA cycle enzymes. 2) Use of extremophile genes to optimize fatty acid production in microalgae for biofuel production. Algal oils are an ideal feedstock for biofuels production, offering very high production density and the ability to use marginal water (municipal waste, brackish water, etc.) and reuse CO2 emitted from coal-fired power plants. However, there are a number of technical challenges associated with efficiently exploiting algae's inherent advantages as a feedstock. One such challenge is that lipid synthesis in microalgae is a highly regulated process than can limit the accumulation of algal lipids. Fatty acid synthesis (FAS) is a highly conserved process in microalgae, archaea and bacteria, in which the pathway that functions in chloroplastic FAS in microalgae also functions similarly in bacteria and archaea. The first committed step in microalgal FAS is the conversion of acetyl CoA to malonyl CoA by acetyl CoA carboxylase (ACCase). This step is also the primary regulated and rate-limiting step for FAS in microalgae, so an increase in ACCase activity should result in increased FAS. Indeed, increased levels of ACCase expression in bacterial systems lead to elevated rates of FA production. Therefore, as a means to overcome transcriptional repression and alleviate feedback inhibition of microalgal ACCase, we propose to express select archaeal and bacterial ACCase genes in the target Dunaliella strain to maximize its FAS. In addition, expression of thioesterases relieves acyl-ACP inhibition of ACCase and allowed increased FAS even under otherwise FASinhibiting conditions. We will also express a type Ithioesterase from C. salexigens (Csal_2620) in Dunaliella in an effort to further limit feedback inhibition of its ACCase. In this reporting period, we were able to 1) demonstrate recombinant expression and activity of the halophilic bacterial ACCase from Chromohalobacter salexigens that will be expressed in Dunaliella, 2) demonstrate recombinant expression and biochemically characterize C. salexigens TesA and show that it can effectively convert acyl-coAs to free fatty acids and 3) generate a C. salexigens TesA expression construct with which to transform Dunaliella. 3) Use of Immobilized Clostridium ljungdahlii in Engineered Biofilms for Synthesis Gas Fermentation to Liquid Fuels. Our model synthesis gas fermentation system is a novel combination of the well-established biofiltration process and the immobilization methods developed by our research group. The proposed activities will use C. ljungdahlii synthesis gas fermentation to define the physiological state of the immobilized cells in our gas phase reactor, characterizing reactivity and response to the most significant trace inhibitors in synthesis gas. In this reporting period we were able to 1) generate and analyze the genome sequence of our wildtype and an improved lab derived strain (OTA1) of Clostridium ljungdahlii, 2) use the genome sequence analysis to identify the mutations that exist in the OTA1 genome which may confer its improved ethanol production in comparison to wildtype C. ljungdahlii, 3) conduct growth, fermentation, enzyme assay and RNA seq analysis of wildtype C. ljungdahlii exposed to low oxygen (0-8%) to evaluate the metabolic response of C. ljungdahlii to oxygen and to determine why oxygen exposure enhances ethanol production by C. ljungdahlii. 4) Application of Extremophile Enzymes for Organophosphorus Compound Detoxification. In the past year we have successfully expressed P. horikoshii prolidase in the microalgae Nannochloropsis oceanica and are currently biochemically characterizing the algal expressed prolidase. 5) Use of reactive oxygen detoxification enzymes from the hyperthermophilic archaeon Pyrococcus furiosus to generate transgenic plants with increased tolerance to harsh environmental conditions. In this reporting period we have introduced the P. furiosus antioxidant enzyme into cyanobacteria and dogwood and have evaluated its efficacy in providing protection against environmental stress. Both transgenic cyanobacteria and dogwood appear to benefit from SOR expression.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Somalinga V , Buhrman G, Arun A, Rose RB, Grunden AM. 2016. A High-Resolution Crystal Structure of a Psychrohalophilic AlphaCarbonic Anhydrase from Photobacterium profundum Reveals a Unique Dimer Interface. PLOS ONE. 11:12 DOI. 10.1371/journal.pone.0168022
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Whitham JM, Schulte MJ, Bobay BG, Bruno-Barcena JM, Chinn MS, Flickinger MC, Pawlak JJ, Grunden AM. 2016. Characterization of Clostridium ljungdahlii OTA1: a non-autotrophic hyper ethanol-producing strain. Appl. Microbiol. Biotechnol. DOI:10.1007/s00253-016-7978-6
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mathews SL, Smithson CE, Grunden AM. 2016. Purification and characterization of a recombinant laccase-like multi-copper oxidase from Paenibacillus glucanolyticus SLM1. J Appl. Microbiol. 2016 Nov;121(5):1335-1345. doi: 10.1111/jam.13241.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kay KL, Breidt F, Fratamico PM, Baranzoni GM, Kim GH, Grunden AM, Oh DH. 2017. Escherichia coli O157:H7 Acid Sensitivity Correlates with Flocculation Phenotype during Nutrient Limitation. Front Microbiol. 8:1404. doi: 10.3389/fmicb.2017.01404. eCollection 2017
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Karam AL, McMillan CC, Lai YC, de Los Reyes FL, Sederoff HW, Grunden AM, Ranjithan RS, Levis JW, Ducoste JJ. 2017. Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests. J Vis Exp. 14;(124). doi: 10.3791/55545.
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: 1. Somalinga V, Klemmer H, Arun A, Wapshott H, Mathews S, Grunden AM. 2018. Cloning, Over-Expression, and Purification of Carbonic Anhydrase from an Extremophilic Bacterium: An Introduction to Advanced Molecular Biology. The American Biology Teacher, 80:29-34.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: 2. Duckworth OW, Andrews MY, Cubeta MA, Grunden AM, Ojiambo PS. 2017. Revisiting Graduate Student Training to Address Agricultural and Environmental Societal Challenge. Agric. Environ. Lett. 2:170019, doi:10.2134/ael2017.06.0019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Integrated Molecular Plant Systems Research at North Carolina State University Grunden, AM 2017 NSF REU Synthetic Biology Presentation Raleigh North Carolina United States
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Identification of Microorganisms from Unique Environments for Application to Agriculture and Biotechnology Grunden, AM Novozymes Site Visit Raleigh North Carolina United States
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Identification and Characterization of Extremophilic Proteins for Microalgal Bioengineering Somalinga V, Grunden AM Biology Seminar at University College of the North in Northern Manitoba Manitoba N/A Canada
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Enhancing Stress Tolerance in Cyanobacteria Kitchener R, Grunden AM Biology Seminar at Campbell University Buies Creek North Carolina United States
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Two-step enzymatic conversion of algal triacylglycerides to hydrocarbons Greenstein J, Wapshott H, Hamilton P, Grunden AM Frontiers in Biorefining Conference St Simon's Island Georgia United States
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grunden, AM. 2017. Increasing Photosynthetic CO2 capture in Camelina sativa with a Synthetic Carbon Fixation Cycle Composed of Selected Microbial Enzymes. Microbiology Seminar Series. University of Georgia February 16, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grunden, AM. Engineering a Better Bioenergy Crop: Development of a Synthetic Carbon Fixation Cycle for Use in C3 Plants. Biology Seminar Series, Univresity of North Carolina, Chapel Hill.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Our research efforts principally benefit the Department of Energy, the National Science Foundation the Department of Agriculture and the Department of Defense, as our research provides important information about development of an effective auxiliary carbon fixation pathway for use in biofuel crop plants, development of algal strains with improved oil and co-product production and biodecontamination systems for detoxification of organophosphorus-based chemical weapons. Our research efforts have also served as a training platform for providing mentored research experience to postdoctoral scholars, as well as graduate, undergraduate, and high school students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported training of twopostdoctoral scholars, threegraduate students, three undergraduates and 4high school students. How have the results been disseminated to communities of interest?Our project results have been disseminated through public lectures, research symposia presentations, and published research articles. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will undertake the following activities as indicated for each of our major project goals: Goal 1: we will 1) finish the in vitro characterization of our crTCA cycle and will submit our findings for publication, 2) will characterize camelina sativa lines that have been transformed with both of the crTCA cycle expression constructs, and 3) will evaluate the effect of the crTCA cycle expression on plant biomass and oilseed production. Goal 2: we will 1) transform the microalgae Dunaliella or other sutiable microalgae with the TesA thioesterase from the halophilic bacterium C. salexigens to improve fatty acid synthesis in the transgenic algae, 2) will transform Dunaliella with industrial extremozymes to provide high value co-product production in the algae, and 3) will identify and characterize enzymes that can be used for nutrient recycle for in a photobioreactor system. Goal 3: we will 1) determine which of the identified mutants in the improved OTA1 C. ljungdahlii strain are responsible for its enhanced ethanol production. Goal 4: we will characterize the extremophile OP degradation enzymes we have expressed in our algae production system and compare the the activity of the algae expressed enzymes to bacterial expressed versions. Goal 5: we will further characterize the SOR expressing cyanobacteria and dogwood and report our findings in journal articles.

Impacts
What was accomplished under these goals? Project Justification/Impact: This project is focused on using extremophiles for biotechnical applications for improved biofuel production, bioremediation and crop improvement. Extremophiles are microorganisms that are capable of thriving in diverse extreme environmental conditions and as a result these microbes have genes/proteins which can have attributes that are valuable to the development of novel biotechnological applications. Currently there are biologically imposed limitations to plant metabolism that constrain plant productivity, and thereby the amount of biofuel that can be generated from these plants. Several of the goals of this research (Obj. 1, 2, and 5) are targeted at improving plant productivity using select extremophile genes. Specifically, we are addressing the limitations of plant-based carbon fixation by designing a new carbon fixation cycle that can be used in plants in conjunction with the traditional Calvin-Benson cycle to convert atmospheric carbon dioxide to plant biomass and biofuel precursor molecules (Objective 1). In addition, extremophile genes are being used to enhance carbon dioxide uptake and nutrient recycle in algal biofuel systems (Obj. 2) and to decontaminate organophosphate based pesticides and chemical warfare agents (Obj. 4. We are also introducing an antioxidant gene coding for superoxide reductase (SOR) into plants to improve the ability of plants to cope with environmental stresses such as temperature, drought, metal, radiation and pathogen stresses (Obj. 5). Biofuels can also be generated from conversion of waste gas streams (CO and CO containing synthesis gas that can be produced from gasification of waste agriculture biomass) using microbial catalysts. We have developed a synthesis gas fermentation platform employing a modified strain of clostridium ljungdahlii and are using gene expression data and fermentation profiles to further improve this system (Obj. 3). Major Project Goals: 1) Generate a novel synthetic carbon fixation cycle composed of select microbial enzymes for use in plant systems to enhance plant biomass and fuel precursor production. Camelina is an excellent biofuel crop system because it grows on poor soils with little water and a short growth period and produces oil-containing seeds with an oil-blend amenable to conversion to transportation fuels, including jet fuels. However,Camelina is a C3 type plant, limiting its carbon fixation capabilities. Objective 1 of this project will generate a new synthetic pathway to increase CO2 assimilation in the chloroplast (condensed, reverse TCA cycle, crTCA cycle) through expression of a suite of genes from plant, bacterial, and archaeal origin. The pathway will be integrated with other transgenes to increase the CO2 concentration inside the chloroplast (CO2 -transporter AQP1), reduce the photorespiratory energy loss (bacterial bypass), and increase photosynthetic efficiency by reducing reactive oxygen species (superoxide reductase). In this reporting period we were able to 1) demonstrate the in vitro function of the full cycle using LC-MS and activity assays, 2) generate two constructs, one which contains the first two enzymes of the cycle and the second which expresses the last three enzymes of the cycle for transformation of Camelina sativa, 3) use transient tobacco transformation to demonstrate in planta activity of four of the five crTCA cycle enzymes. 2) Use of extremophile genes to optimize fatty acid production in microalgae for biofuel production. Algal oils are an ideal feedstock for biofuels production, offering very high production density and the ability to use marginal water (municipal waste, brackish water, etc.) and reuse CO2 emitted from coal-fired power plants. However, there are a number of technical challenges associated with efficiently exploiting algae's inherent advantages as a feedstock. One such challenge is that lipid synthesis in microalgae is a highly regulated process than can limit the accumulation of algal lipids. Fatty acid synthesis (FAS) is a highly conserved process in microalgae, archaea and bacteria, in which the pathway that functions in chloroplastic FAS in microalgae also functions similarly in bacteria and archaea. The first committed step in microalgal FAS is the conversion of acetyl CoA to malonyl CoA by acetyl CoA carboxylase (ACCase). This step is also the primary regulated and rate-limiting step for FAS in microalgae, so an increase in ACCase activity should result in increased FAS. Indeed, increased levels of ACCase expression in bacterial systems lead to elevated rates of FA production. Therefore, as a means to overcome transcriptional repression and alleviate feedback inhibition of microalgal ACCase, we propose to express select archaeal and bacterial ACCase genes in the target Dunaliella strain to maximize its FAS. In addition, expression of thioesterases relieves acyl-ACP inhibition of ACCase and allowed increased FAS even under otherwise FASinhibiting conditions. We will also express a type I thioesterase from C. salexigens (Csal_2620) in Dunaliella in an effort to further limit feedback inhibition of its ACCase. In this reporting period, we were able to 1) demonstrate recombinantexpression and activity of the halophilic bacterial ACCase from Chromohalobacter salexigens that will be expressed in Dunaliella, 2) demonstrate recombinant expression and biochemically characterize C. salexigens TesA and show that it can effectively convert acyl-coAs to free fatty acids and 3) generate a C. salexigens TesA expression construct with which to transform Dunaliella. 3) Use of Immobilized Clostridium ljungdahlii in Engineered Biofilms for Synthesis Gas Fermentation to Liquid Fuels.Our model synthesis gas fermentation system is a novel combination of the well-established biofiltration process and the immobilization methods developed by our research group. The proposed activities will use C. ljungdahlii synthesis gas fermentation to define the physiological state of the immobilized cells in our gas phase reactor, characterizing reactivity and response to the most significant trace inhibitors in synthesis gas. In this reporting period we were able to 1) generate and analyze the genome sequence of our wildtype and an improved lab derived strain (OTA1) of Clostridium ljungdahlii, 2) use the genome sequence analysis to identify the mutations that exist in the OTA1 genome which may confer its improved ethanol production in comparison to wildtype C. ljungdahlii, 3) conduct growth, fermentation, enzyme assay and RNA seq analysis of wildtype C. ljungdahlii exposed to low oxygen (0-8%) to evaluate the metabolic response of C. ljungdahlii to oxygen and to determine why oxygen exposure enhances ethanol production by C. ljungdahlii. 4) Application of Extremophile Enzymes for Organophosphorus Compound Detoxification. In the past year we havesuccessfully expressedP. horikoshiiprolidase in the microalgaeNannochloropsis oceanicaand are currently biochemically characterizing the algal expressed prolidase. 5) Use of reactive oxygen detoxification enzymes from the hyperthermophilic archaeon Pyrococcus furiosus to generate transgenic plants with increased tolerance to harsh environmental conditions. In this reporting period we have introduced the P. furiosus antioxidant enzyme into cyanobacteria and dogwood and have evaluated its efficacy in providing protection against environmental stress. Both transgenic cyanobacteria and dogwood appear to benefit from SOR expression.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Whitham JM, Schulte MJ, Bobay BG, Bruno-Barcena JM, Chinn MS, Flickinger MC, Pawlak JJ, Grunden AM. 2016. Characterization of Clostridium ljungdahlii OTA1: a non-autotrophic hyper ethanol-producing strain. Appl. Microbiol. Biotechnol. DOI:10.1007/s00253-016-7978-6
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mathews SL, Smithson CE, Grunden AM. 2016. Purification and characterization of a recombinant laccase-like multi-copper oxidase from Paenibacillus glucanolyticus SLM1. J Appl. Microbiol. 2016 Nov;121(5):1335-1345. doi: 10.1111/jam.13241.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mathews SL, Pawlak J, Grunden AM. 2016. Draft Genome Sequences of Two Strains of Paenibacillus glucanolyticus with the Ability To Degrade Lignocellulose. Genome Announc. Jun 23;4(3). pii: e00423-16. doi: 10.1128/genomeA.00423-16.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Geng X, Liu X, Ji M, Hoffman W, Xiang Q, Grunden AM. 2016. Enhancing heat tolerance of the little dogwood Cornus canadensis L. f. with introduction of a superoxide reductase gene from the hyperthermophilic archaeon Pyrococcus furiosus. Frontiers in Plants Science- Plant Biotechnology. 7:26, doi: 10.3389/fpls.2016.00026.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mathews SL, Grunden AM, Pawlak JJ. 2016. Degradation of Lignocellulose and Lignin by Paenibacillus glucanolyticus.. International Biodeterioration & Biodegradation. 110:79-86, doi:10.1016/j.ibiod.2016.02.012.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Whitham JM, Pawlak JJ, Grunden AM. 2016. Clostridium ljungdahlii: a Review of the Development of an Industrial Biocatalyst. Current Biotechnology. 5(1): 54-70.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Savage AM, Hills J, Driscoll K, Fergus DJ, Grunden AM, Dunn RR. 2016. Microbial diversity of extreme habitats in human homes. PeerJ. Sep 13;4:e2376. doi: 10.7717/peerj.2376.
  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Kay KL, Breidt F, Fratamico PM, Baranzoni GM, Kim GH, Grunden AM and Oh DH Escherichia coli O157:H7 Acid Sensitivity is Distinguished by the Curli Phenotype during Nutrient Limitation. Frontiers in Microbiology
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Two-step enzymatic conversion of algal triacylglycerides to hydrocarbons Greenstein J, Wapshott H, Hamilton P, Grunden AM Frontiers in Biorefining Conference St Simon's Island Georgia United States Start Date: 11/09/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Crystal structure of an alpha-carbonic anhydrase from a barotolerant psychrohalophile with a novel chloride ion in the dimer interface Buhrman G, Somalinga V, Arun A, Grunden AM and Rose RB 46th Mid-Atlantic Macromolecular Crystallography Meeting Charlottsburg Virginia United States Start Date: 06/08/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Structural and functional characterization of an alpha-carbonic anhydrase from a psychrohalophilic bacterium, Photobacterium profundum Somalinga V, Buhrman G, Arun A, Rose RB and Grunden AM 5th Annual Postdoctoral Research Symposium, North Carolina State University Raleigh North Carolina United States Start Date: 05/17/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Expression of Extremozymes in Biofuel-Producing Cyanobacteria:Two Valuable Applications Kitchener R, Murphree C, Young D, Sederoff H, and Grunden A NCSU Springboard Innovation Forum Raleigh North Carolina United States Start Date: 01/25/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Integrated Molecular Plant Systems Research at North Carolina State University Grunden, AM 2016 NSF REU Synthetic Biology Presentation Raleigh North Carolina United States Start Date: 05/25/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Identification of Microorganisms from Unique Environments for Application to Agriculture and Biotechnology Grunden, AM Novozymes Site Visit Raleigh North Carolina United States Start Date: 06/09/2016
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Somalinga V , Buhrman G, Arun A, Rose RB, Grunden AM. 2016. A High-Resolution Crystal Structure of a Psychrohalophilic ?Carbonic Anhydrase from Photobacterium profundum Reveals a Unique Dimer Interface. Plos ONE. Vol 11. DOI: 10.1371/journal.pone.0168022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Improving Biofuel Production in Marine Microalgae Somalinga V, Grunden AM PDAW NRC: Science on Stage at the North Carolina Museum of Natural Sciences Raleigh North Carolina United States Start Date: 09/20/2016 End Date: 09/24/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Enhancing Stress Tolerance in Cyanobacteria Kitchener R, Grunden AM Chemical and Biological Engineering Biolunch Seminar Series Raleigh North Carolina United States Start Date: 07/27/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Enhancing Stress Tolerance in Cyanobacteria Kitchener R, Grunden AM Biology Seminar at Campbell University Buies Creek North Carolina United States Start Date: 11/28/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Characterization of a putative thermostable fatty acid decarboxylase to improve algal biofuel processing Wapshott H, Grunden AM Chemical and Biological Engineering Biolunch Seminar Series Raleigh North Carolina United States Start Date: 06/08/2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Identification and Characterization of Extremophilic Proteins for Microalgal Bioengineering Somalinga V, Grunden AM Biology Seminar at University College of the North in Northern Manitoba Manitoba N/A Canada Start Date: 05/27/2016


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Our research efforts principally benefit the Department of Energy, the National Science Foundation the Department of Agriculture and the Department of Defense, as our research provides important information about development of an effective auxiliary carbon fixation pathway for use in biofuel crop plants, development of algal strains with improved oil and co-product production and biodecontamination systems for detoxification of organophosphorus-based chemical weapons. Our research efforts have also served as a training platform for providing mentored research experience to postdoctoral scholars, as well as graduate, undergraduate, and high school students. Changes/Problems:I have no changes or problems to discuss for this reporting period. What opportunities for training and professional development has the project provided?The project has supported training of five postdoctoral scholars, five graduate students, three undergraduates and 5 high school students. How have the results been disseminated to communities of interest?Our project results have been disseminated through public lectures, research symposia presentations, and published research articles. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will undertake the following activities as indicated for each of our major project goals: Goal 1: we will 1) finish the in vitro characterization of our crTCA cycle and will submit our findings for publication, 2) transform camelina sativa with both of the crTCA cycle expression constructs, and 3) will evaluate the effect of the crTCA cycle expression on plant biomass and oilseed production. Goal 2: we will 1) transform the microalgae Dunaliella or other sutiable microalgae with the TesA thioesterase from the halophilic bacterium C. salexigens to improve fatty acid synthesis in the transgenic algae, 2) will transform Dunaliella with industrial extremozymes to provide high value co-product production in the algae, and 3) will identify and characterize enzymes that can be used for nutrient recycle for in a photobioreactor system. Goal 3: we will 1) determine which of the identified mutants in the improved OTA1 C. ljungdahlii strain are responsible for its enhanced ethanol production. Goal 4: we will express the extremophile OP degradation enzymes in our algae production system and evaluate the activity of the algae expressed enzymes Goal 5: we will further characterize the SOR expressing cyanobacteria and dogwood and report our findings in journal articles.

Impacts
What was accomplished under these goals? PrProject Justification/Impact: This project is focused on using extremophiles for biotechnical applications for improved biofuel production, bioremediation and crop improvement. Extremophiles are microorganisms that are capable of thriving in diverse extreme environmental conditions and as a result these microbes have genes/proteins which can have attributes that are valuable to the development of novel biotechnological applications. Currently there are biologically imposed limitations to plant metabolism that constrain plant productivity, and thereby the amount of biofuel that can be generated from these plants. Several of the goals of this research (Obj. 1, 2, and 5) are targeted at improving plant productivity using select extremophile genes. Specifically, we are addressing the limitations of plant-based carbon fixation by designing a new carbon fixation cycle that can be used in plants in conjunction with the traditional Calvin-Benson cycle to convert atmospheric carbon dioxide to plant biomass and biofuel precursor molecules (Objective 1). In addition, extremophile genes are being used to enhance carbon dioxide uptake and nutrient recycle in algal biofuel systems (Obj. 2) and to decontaminate organophosphate based pesticides and chemical warfare agents (Obj. 4. We are also introducing an antioxidant gene coding for superoxide reductase (SOR) into plants to improve the ability of plants to cope with environmental stresses such as temperature, drought, metal, radiation and pathogen stresses (Obj. 5). Biofuels can also be generated from conversion of waste gas streams (CO and CO containing synthesis gas that can be produced from gasification of waste agriculture biomass) using microbial catalysts. We have developed a synthesis gas fermentation platform employing a modified strain of clostridium ljungdahlii and are using gene expression data and fermentation profiles to further improve this system (Obj. 3). Major Project Goals: 1) Generate a novel synthetic carbon fixation cycle composed of select microbial enzymes for use in plant systems to enhance plant biomass and fuel precursor production. Camelina is an excellent biofuel crop system because it grows on poor soils with little water and a short growth period and produces oil-containing seeds with an oil-blend amenable to conversion to transportation fuels, including jet fuels. However,Camelina is a C3 type plant, limiting its carbon fixation capabilities. Objective 1 of this project will generate a new synthetic pathway to increase CO2 assimilation in the chloroplast (condensed, reverse TCA cycle, crTCA cycle) through expression of a suite of genes from plant, bacterial, and archaeal origin. The pathway will be integrated with other transgenes to increase the CO2 concentration inside the chloroplast (CO2-transporter AQP1), reduce the photorespiratory energy loss (bacterial bypass), and increase photosynthetic efficiency by reducing reactive oxygen species (superoxide reductase). In this reporting period we were able to 1) demonstrate the in vitro function of the full cycle using LC-MS and activity assays, 2) generate two constructs, one which contains the first two enzymes of the cycle and the second which expresses the last three enzymes of the cycle for transformation of Camelina sativa, 3) use transient tobacco transformation to demonstrate in planta activity of four of the five crTCA cycle enzymes. 2) Use of extremophile genes to optimize fatty acid production in microalgae for biofuel production Algal oils are an ideal feedstock for biofuels production, offering very high production density and the ability to use marginal water (municipal waste, brackish water, etc.) and reuse CO2 emitted from coal-fired power plants. However, there are a number of technical challenges associated with efficiently exploiting algae's inherent advantages as a feedstock. One such challenge is that lipid synthesis in microalgae is a highly regulated process than can limit the accumulation of algal lipids. Fatty acid synthesis (FAS) is a highly conserved process in microalgae, archaea and bacteria, in which the pathway that functions in chloroplastic FAS in microalgae also functions similarly in bacteria and archaea. The first committed step in microalgal FAS is the conversion of acetyl CoA to malonyl CoA by acetyl CoA carboxylase (ACCase). This step is also the primary regulated and rate-limiting step for FAS in microalgae, so an increase in ACCase activity should result in increased FAS. Indeed, increased levels of ACCase expression in bacterial systems lead to elevated rates of FA production. Therefore, as a means to overcome transcriptional repression and alleviate feedback inhibition of microalgal ACCase, we propose to express select archaeal and bacterial ACCase genes in the target Dunaliella strain to maximize its FAS. In addition, expression of thioesterases relieves acyl-ACP inhibition of ACCase and allowed increased FAS even under otherwise FAS-inhibiting conditions. We will also express a type I thioesterase from C. salexigens (Csal_2620) in Dunaliella in an effort to further limit feedback inhibition of its ACCase. In this reporting period, we were able to 1) demonstrate recombinant expression and activity of the halophilic bacterial ACCase from Chromohalobacter salexigens that will be expressed in Dunaliella, 2) demonstrate recombinant expression and biochemically characterize C. salexigens TesA and show that it can effectively convert acyl-coAs to free fatty acids and 3) generate a C. salexigens TesA expression construct with which to transform Dunaliella. 3) Use of Immobilized Clostridium ljungdahlii in Engineered Biofilms for Synthesis Gas Fermentation to Liquid Fuels Our model synthesis gas fermentation system is a novel combination of the well-established biofiltration process and the immobilization methods developed by our research group. The proposed activities will use C. ljungdahlii synthesis gas fermentation to define the physiological state of the immobilized cells in our gas phase reactor, characterizing reactivity and response to the most significant trace inhibitors in synthesis gas. In this reporting period we were able to 1) generate and analyze the genome sequence of our wildtype and an improved lab derived strain (OTA1) of Clostridium ljungdahlii, 2) use the genome sequence analysis to identify the mutations that exist in the OTA1 genome which may confer its improved ethanol production in comparison to wildtype C. ljungdahlii, 3) conduct growth, fermentation, enzyme assay and RNA seq analysis of wildtype C. ljungdahlii exposed to low oxygen (0-8%) to evaluate the metabolic response of C. ljungdahlii to oxygen and to determine why oxygen exposure enhances ethanol production by C. ljungdahlii. 4 4) Application of Extremophile Enzymes for Organophosphorus Compound Detoxification In the past year we have extended our studies by biochemically evaluating a related prolidase from the hyperthermophile Pyrococcus horikoshii, and we have shown that this prolidase, Ph1-prol, has even greater catalytic efficiency than the P. furiosus prolidase, and therefore, we have also bioengineered this enzyme to generate mutant enzymes that have good activity over a broad range of temperatures. The work that we have done to generate a highly active recombinant versions of both the P. furiosus and P. horikoshii prolidase enzymes that are active over a broad range of temperatures. 5) Use of reactive oxygen detoxification enzymes from the hyperthermophilic archaeon Pyrococcus furiosus to generate transgenic plants with increased tolerance to harsh environmental conditions. In this reporting period we have introduced the P. furiosus antioxidant enzyme into cyanobacteria and dogwood and have evaluated its efficacy in providing protection against environmental stress. Both transgenic cyanobacteria and dogwood appear to benefit from SOR expression.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Whitham JM, Tirado-Acevedo O, Chinn MS, Pawlak JJ, Grunden AM. 2015. Metabolic Response of Clostridium Ljungdahlii to Oxygen Exposure. Appl Environ Microbiol. 2015 Dec 15;81(24):8379-91. doi: 10.1128/AEM.02491-15.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ji ML, Grunden AM. 2015. Cloning, Purification and Characterization of Recombinant Glutathione Reductase from the Psychrophilic Antarctic Bacterium, Colwellia psychrerythraea. Extremophiles. DOI 10.1007/s00792-015-0762-1
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Aslett LD, Ji ML, Li K, Lin X, Smith CM, Wapshott HL, Dalal J, Bobay B, Sederoff H, Xie D, and Grunden AM, Increasing photosynthetic CO2 capture in Camelina with a synthetic carbon fixation cycle composed of select microbial enzymes, Meeting of the North Carolina Branch of American Society for Microbiology, North Carolina State University, Raleigh, North Carolina 3rd October 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kay K, Fratamico P, Grunden AM, Oh D-H, Breidt F. Investigation of Carbon Storage Regulation Network (csr genes) and Phenotypic Differences Between Acid Sensitive and Resistant Escherichia coli O157:H7 Strains. 115th General Meeting of the American Society for Microbiology. New Orleans, LA May 30  June 2, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Mathews SL, Pawlak J, Grunden AM. Degradation of Lignin and Lignin-Related Compounds by Paenibacillus glucanolyticus. Meeting of the North Carolina Branch of American Society for Microbiology, North Carolina State University, Raleigh, North Carolina 3rd October 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Dalal, J., Yalamanchili, R., Hovary,C.L., Ji,M., Rodriguez-Welsh,M., Aslett,D., Ganapathy,S., Grunden,A., Sederoff, H. and Qu,R. 2015. A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for the genetic transformation of plants. Plasmid, 81:55-62. doi: 10.1016/j.plasmid.2015.06.003
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Utturkar S, Klingeman DM, Bruno-Barcena JM, Chinn MS, Grunden AM, K�pke M, Brown SD. 2015. Sequence Data for Clostridium autoethanogenum using Three Generations of Sequencing Technologies. Scientific Data 04/2015; 2:150014. DOI: 10.1038/sdata.2015.14
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Mathews SL, Pawlak JJ, Grunden AM. 2015. Bacterial Biodegradation and Bioconversion of Industrial Lignocellulosic Streams. Appl. Microbial. Biotechnol. 99(7):2939-2954.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Aslett LD, Ji ML, Li K, Lin X, Smith CM, Wapshott HL, Dalal J, Bobay B, Sederoff H, Xie D, Grunden AM, Jet Fuel from Camelina. DOE Innovation Summit, Gaylord Convention Center, Washington, D.C. February, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Mathews SL, Hamilton P, Dunn R, Grunden AM. Industrial Enzymes from the Microbiome of Household Insects. NSF I-CORPS Kickoff meeting. Los Angeles, CA. October 26, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zering, KD, Natelson RH, Mayer ML, Grunden, AM, Sederoff H. NC-CAB2 - The North Carolina Consortium for Algae Bioenergy and Bioproducts. State Energy Conference Think and Do: Mapping a Sustainable Future for Energy in North Carolina. Raleigh, McKimmon Center, April 21-22, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Mathews SL, Hamilton P, Dunn R, Grunden AM. Industrial Enzymes from the Microbiome of Household Insects. NSF I-CORPS Lesson Learned meeting. Los Angeles, CA. December 13, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Smith, C.M., Aslett, D., Ji, M., Li, K., Lin, X., Wapshott, H., Bobay, B., Xie, D., Sederoff, H., and Grunden, A.M. Development of a Microbe-Derived, Synthetic Condensed Reverse TCA Cycle to Improve Carbon Fixation in the Seed Oil Crop Camelina sativa. Genetic Engineering at NCSU Symposium, Raleigh, NC, Apr. 28, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zering KD, Natelson RH, Mayer ML, Grunden, AM, and Sederoff H. Economics and Commercialization of Camelina Biofuels in North Carolina. State Energy Conference: Think and Do: Mapping a Sustainable Future for Energy in North Carolina, Raleigh, McKimmon Center, April 21-22, 2015.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Our research efforts principally benefit the Department of Energy, the National Science Foundation the Department of Agriculture and the Department of Defense, as our research provides important information about development of an effective auxiliary carbon fixation pathway for use in biofuel crop plants, development of algal strains with improved oil and co-product production and biodecontamination systems for detoxification of organophosphorus-based chemical weapons. Our research efforts have also served as a training platform for providing mentored research experience to graduate and undergraduate students. Changes/Problems: I have no changes or problems to describe in this reporting period. What opportunities for training and professional development has the project provided? The project has supported training of four postdoctoral scholars, six graduate students, three undergraduates and 4 high school students. How have the results been disseminated to communities of interest? Our project results have been disseminated through public lectures, research symposia presentations, published research articles and books. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period we will undertake the following activities as indicated for each of our major project goals: Goal 1: we will 1) finish the in vitro characterization of our crTCA cycle and will submit our findings for publication, 2) transform camelina sativa with both of the crTCA cycle expression constructs, and 3) will evaluate the effect of the crTCA cycle expression on plant biomass and oilseed production. Goal 2: we will 1) transform the microalgae Dunaliella with the TesA thioesterase from the halophilic bacterium C. salexigens to improve fatty acid synthesis in the transgenic algae, 2) will transform Dunaliella with industrial extremozymes to provide high value co-product production in the algae, and 3) will identify and characterize enzymes that can be used for nutrient recycle for in a photobioreactor system. Goal 3: we will 1) determine which of the identified mutants in the improved OTA1 C. ljungdahlii strain are responsible for its enhanced ethanol production. Goal 4: we will express the extremophile OP degradation enzymes in our algae production system and evaluate the activity of the algae expressed enzymes Goal 5: we will further characterize the SOR expressing cyanobacteria and dogwood and report our findings in journal articles.

Impacts
What was accomplished under these goals? Project Justification/Impact This project is focused on using extremophiles for biotechnical applications for improved biofuel production, bioremediation and crop improvement. Extremophiles are microorganisms that are capable of thriving in diverse extreme environmental conditions and as a result these microbes have genes/proteins which can have attributes that are valuable to the development of novel biotechnological applications. Currently there are biologically imposed limitations to plant metabolism that constrain plant productivity, and thereby the amount of biofuel that can be generated from these plants. Several of the goals of this research (Obj. 1, 2, and 5) are targeted at improving plant productivity using select extremophile genes. Specifically, we are addressing the limitations of plant-based carbon fixation by designing a new carbon fixation cycle that can be used in plants in conjunction with the traditional Calvin-Benson cycle to convert atmospheric carbon dioxide to plant biomass and biofuel precursor molecules (Objective 1). In addition, extremophile genes are being used to enhance carbon dioxide uptake and nutrient recycle in algal biofuel systems (Obj. 2) and to decontaminate organophosphate based pesticides and chemical warfare agents (Obj. 4. We are also introducing an antioxidant gene coding for superoxide reductase (SOR) into plants to improve the ability of plants to cope with environmental stresses such as temperature, drought, metal, radiation and pathogen stresses (Obj. 5). Biofuels can also be generated from conversion of waste gas streams (CO and CO2containing synthesis gas that can be produced from gasification of waste agriculture biomass) using microbial catalysts. We have developed a synthesis gas fermentation platform employing a modified strain ofclostridium ljungdahliiand are using gene expression data and fermentation profiles to further improve this system (Obj. 3). Major Project Goals: 1) Generate a novel synthetic carbon fixation cycle composed of select microbial enzymes for use in plant systems to enhance plant biomass and fuel precursor production. Camelina is an excellent biofuel crop system because it grows on poor soils with little water and a short growth period and produces oil-containing seeds with an oil-blend amenable to conversion to transportation fuels, including jet fuels. However, Camelina is a C3 type plant, limiting its carbon fixation capabilities. Objective 1 of this project will generate a new synthetic pathway to increase CO2 assimilation in the chloroplast (condensed, reverse TCA cycle, crTCA cycle) through expression of a suite of genes from plant, bacterial, and archaeal origin. The pathway will be integrated with other transgenes to increase the CO2 concentration inside the chloroplast (CO2-transporter AQP1), reduce the photorespiratory energy loss (bacterial bypass), and increase photosynthetic efficiency by reducing reactive oxygen species (superoxide reductase). In this reporting period we were able to 1) demonstrate the in vitro function of the full cycle using LC-MS and activity assays, 2) generate two constructs, one which contains the first two enzymes of the cycle and the second which expresses the last three enzymes of the cycle for transformation of Camelina sativa, 3) use transient tobacco transformation to demonstrate in planta activity of four of the five crTCA cycle enzymes. 2) Use of extremophile genes to optimize fatty acid production in microalgae for biofuel production Algal oils are an ideal feedstock for biofuels production, offering very high production density and the ability to use marginal water (municipal waste, brackish water, etc.) and reuse CO2 emitted from coal-fired power plants. However, there are a number of technical challenges associated with efficiently exploiting algae's inherent advantages as a feedstock. One such challenge is that lipid synthesis in microalgae is a highly regulated process than can limit the accumulation of algal lipids. Fatty acid synthesis (FAS) is a highly conserved process in microalgae, archaea and bacteria, in which the pathway that functions in chloroplastic FAS in microalgae also functions similarly in bacteria and archaea. The first committed step in microalgal FAS is the conversion of acetyl CoA to malonyl CoA by acetyl CoA carboxylase (ACCase). This step is also the primary regulated and rate-limiting step for FAS in microalgae, so an increase in ACCase activity should result in increased FAS. Indeed, increased levels of ACCase expression in bacterial systems lead to elevated rates of FA production. Therefore, as a means to overcome transcriptional repression and alleviate feedback inhibition of microalgal ACCase, we propose to express select archaeal and bacterial ACCase genes in the target Dunaliella strain to maximize its FAS. In addition, expression of thioesterases relieves acyl-ACP inhibition of ACCase and allowed increased FAS even under otherwise FAS-inhibiting conditions. We will also express a type I thioesterase from C. salexigens (Csal_2620) in Dunaliella in an effort to further limit feedback inhibition of its ACCase. In this reporting period, we were able to 1) demonstrate recombinant expression and activity of the halophilic bacterial ACCase from Chromohalobacter salexigens that will be expressed in Dunaliella, 2) demonstrate recombinant expression and biochemically characterize C. salexigens TesA and show that it can effectively convert acyl-coAs to free fatty acids and 3) generate a C. salexigens TesA expression construct with which to transform Dunaliella. 3) Use of Immobilized Clostridium ljungdahlii in Engineered Biofilms for Synthesis Gas Fermentation to Liquid Fuels Our model synthesis gas fermentation system is a novel combination of the well-established biofiltration process and the immobilization methods developed by our research group. The proposed activities will use C. ljungdahlii synthesis gas fermentation to define the physiological state of the immobilized cells in our gas phase reactor, characterizing reactivity and response to the most significant trace inhibitors in synthesis gas. In this reporting period we were able to 1) generate and analyze the genome sequence of our wildtype and an improved lab derived strain (OTA1) of Clostridium ljungdahlii, 2) use the genome sequence analysis to identify the mutations that exist in the OTA1 genome which may confer its improved ethanol production in comparison to wildtype C. ljungdahlii, 3) conduct growth, fermentation, enzyme assay and RNA seq analysis of wildtype C. ljungdahlii exposed to low oxygen (0-8%) to evaluate the metabolic response of C. ljungdahlii to oxygen and to determine why oxygen exposure enhances ethanol production by C. ljungdahlii. 4) Application of Extremophile Enzymes for Organophosphorus Compound Detoxification In the past year we have extended our studies by biochemically evaluating a related prolidase from the hyperthermophile Pyrococcus horikoshii, and we have shown that this prolidase, Ph1-prol, has even greater catalytic efficiency than the P. furiosus prolidase, and therefore, we have also bioengineered this enzyme to generate mutant enzymes that have good activity over a broad range of temperatures. The work that we have done to generate a highly active recombinant versions of both the P. furiosus and P. horikoshii prolidase enzymes that are active over a broad range of temperatures. 5) Use of reactive oxygen detoxification enzymes from the hyperthermophilic archaeon Pyrococcus furiosus to generate transgenic plants with increased tolerance to harsh environmental conditions. In this reporting period we have introduced the P. furiosus antioxidant enzyme into cyanobacteria and dogwood and have evaluated its efficacy in providing protection against environmental stress. Both transgenic cyanobacteria and dogwood appear to benefit from SOR expression.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mathews SL, Pawlak JJ, Grunden AM. 2014. Isolation of Paenibacillus glucanolyticus from Pulp Mill Sources with Potential to Deconstruct Pulping Waste. Bioresource Technology. 164: 100105.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Im YJ, Smith C, Phillippy B, Strand D, Kramer D, Grunden AM, Boss WF. 2014. Increasing phosphatidylinositol (4,5) bisphosphate biosynthesis affects basal signaling and chloroplast metabolism in Arabidopsis thaliana. Plants. 3:27-57. doi:10.3390/plants3010027
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Schreck S, Killens-Cade R, Grunden AM. 2014. Characterization of a Halophilic Acyl-CoA Thioesterase from Chromohalobacter salexigens for Use in Biofuel Production. Current Biotechnology. 2: 275-283.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Killens R, Turner R, McInnes C, Grunden AM. 2014. Characterization of a recombinant Metallosphaera sedula carboxylesterase for use in algal-based biofuel production. Advances in enzyme Research. 2:1-13.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Killens-Cade RR, Kitchener RL, Mathews SL, Schreck S, Ji ML, Turner R, MacInnes C, Gunden AM. 2014. Production of extremophilic proteins using Escherichia coli-based expression systems. In Basic Methods in Protein Purification and Analysis. iConcept Press, Ltd.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Mathews SL, Pawlak JJ, Grunden AM. 2015. Bacterial Biodegradation and Bioconversion of Industrial Lignocellulosic Streams. Submitted to Appl. Microbial. Biotechnol.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Whitham J., Tirado-Acevedo O, Chinn MS, Pawlak JJ, Grunden AM. 2015. Metabolic response of Clostridium ljungdahlii to oxygen exposure. App. Environ. Microbiol. Under review.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Killens-Cade R. 2014. Biochemical Characterization of Extremophile Fatty Acid Metabolism Enzymes for Use in Algal-Based Biofuel Production. North Carolina State University
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Smith, C.M., Aslett, D., Ji, M., Li, K., Lin, X., Dalal, J., Wapshott, H., Bobay, B., Xie, D., Sederoff, H., and Grunden, A.M. Development of a Microbe-Derived, Synthetic Condensed Reverse TCA Cycle to Improve Carbon Fixation in the Seed Oil Crop Camelina sativa. Poster presented at: American Society for Microbiology, General Meeting; 2014 May 17-20; Boston, MA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Smith, C.M., Aslett, D., Ji, M., Li, K., Lin, X., Dalal J., Wapshott, H., Bobay, B., Xie, D., Sederoff, H., and Grunden, A.M. Development of a Microbe-Derived, Synthetic Condensed Reverse TCA Cycle to Improve Carbon Fixation in the Seed Oil Crop Camelina sativa. Poster presented at: Duke Symposium on Microbial Systems Physiology; 2014 October 10; Durham, NC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mikyoung Ji, Callie Barnwell, and Amy Grunden Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium, Colwellia psychrerythraea, 9th Annual Duke Systems Biology Symposium -Microbial Systems Physiology, Oct. 2014, Duke university, Durham, NC
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mathews, S.L., Pawlak, J.J., Grunden, A.M. "Biodegradation and Bioconversion of Pulping Waste by Paenibacillus glucanolyticus", ASM general meeting, Poster, Boston, MA May 2014
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mathews, S.L., Pawlak, J.J., Grunden, A.M. "Biodegradation and Bioconversion of Pulping Waste by Paenibacillus glucanolyticus", Duke Systems Biology Symposium, Poster, Durham NC October 2014
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Whitham, J. M., O. Tirado-Acevedo, M. S. Chinn, J. J. Pawlak, A. M. Grunden. (2014, May). Metabolic Response of Clostridium ljungdahlii to Oxygen Exposure. Poster session presented at the American Society of Microbiology General Meeting at the Boston Convention and Exhibition Center in Boston, MA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Whitham, J. M., O. Tirado-Acevedo, M. S. Chinn, J. J. Pawlak, A. M. Grunden. (2014, May). Metabolic Response of Clostridium ljungdahlii to Oxygen Exposure. Poster session presented at the 1st Annual RTP Chemical Biology and Biotechnology Symposium at GlaxoSmithKline, RTP, NC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sederoff, H., Grunden A., Xie, D., Qu, R., Veal, M., Zering, K., Roberts, B. Metabolic Engineering for Yield and Fuel.Poster presentation at the DOE ARPA-E Innovation Summit Showcase. February 24-26, 2014. National Harbour, MD.