Robert Kelly
Bio
We are interested in the genomics, physiology, enzymology and biotechnological potential of microorganisms that thrive in extreme environments, i.e., extremes in temperature, pressure, pH, ionic strength, etc. Our primary focus is on extremely thermophilic microorganisms, which are isolated from geothermal sites and volcanic regions and typically have optimal growth temperatures above 70°C. Because of the high temperatures at which these bacteria and archaea can be cultured, they produce highly thermostable enzymes that hold promise as biocatalysts. Metabolic pathways encoded in the genomes of extreme thermophiles have great potential for technologically important biotransformations. Molecular genetics systems have recently become available for several extreme thermophiles, thereby creating opportunities for metabolic engineering and synthetic biology at high temperatures.
Our research efforts are aimed at the interface between biology and engineering. We have addressed issues of fundamental importance in understanding the bioenergetics, biochemistry, physiology and genomics of extreme thermophiles. These studies have given rise to a number of technologically important developments related to bienergy and biofuels, recovery of base, precious and strategic metals from ores, and industrial biocatalysis. Students involved in this research should expect to develop expertise in biochemistry, biophysics, microbiology, molecular biology, and genomics to complement their training in biomolecular engineering.
Research Areas – Biomolecular Engineering, Biocatalysis at Extremely High Temperatures, Microbial Physiology, Functional Genomics, Bioenergy and Biofuels.
Publications
- Complete genome sequence for the extremely thermophilic bacterium Anaerocellum danielii (DSM:8977) , MICROBIOLOGY RESOURCE ANNOUNCEMENTS (2024)
- Complete genome sequence for the thermoacidophilic archaeon Metallosphaera sedula (DSM:5348) , MICROBIOLOGY RESOURCE ANNOUNCEMENTS (2024)
- Extremely thermoacidophilic archaea for metal bioleaching: What do their genomes tell Us? , BIORESOURCE TECHNOLOGY (2024)
- Complete Genome Sequences of Caldicellulosiruptor acetigenus DSM 7040, Caldicellulosiruptor morganii DSM 8990 (RT8.B8), and Caldicellulosiruptor naganoensis DSM 8991 (NA10) , MICROBIOLOGY RESOURCE ANNOUNCEMENTS (2023)
- Complete Genome Sequences of Two Thermophilic Indigenous Bacteria Isolated from Wheat Straw, Thermoclostridium stercorarium subsp. Strain RKWS1 and Thermoanaerobacter sp. Strain RKWS2 , MICROBIOLOGY RESOURCE ANNOUNCEMENTS (2023)
- Complete genome sequence for the thermoacidophilic archaeon Sulfuracidifex (f. Sulfolobus) metallicus DSM 6482 , MICROBIOLOGY RESOURCE ANNOUNCEMENTS (2023)
- Fermentative conversion of unpretreated plant biomass: A thermophilic threshold for indigenous microbial growth , BIORESOURCE TECHNOLOGY (2023)
- Innovating Life Sciences Laboratory Training: Molecular Biology Laboratory Education Modules (MBLEMs) as a Model for Advanced Training at Diverse Institutions , JOURNAL OF BIOLOGICAL CHEMISTRY (2023)
- Interplay between transcriptional regulators and VapBC toxin-antitoxin loci during thermal stress response in extremely thermoacidophilic archaea , ENVIRONMENTAL MICROBIOLOGY (2023)
- Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95 degrees C Driven by Carbon Monoxide Oxidation , APPLIED AND ENVIRONMENTAL MICROBIOLOGY (2023)
Grants
Training in molecular biotechnology is essential for an expanding list of disciplines that have found modern biology-based skills of critical importance in pursuing research goals in areas ranging from biochemistry to chemical engineering to plant biology. Recognizing this, NC State University has created a core education facility that serves campus-wide needs for graduate students requiring laboratory-based training in aspects of modern biology. This not only facilitates completion of the students? dissertation research, but also lays the basis for career opportunities in academic, government and industrial research settings. Using this campus educational resource as a framework, NC State University proposes to continue a graduate level training program in molecular biotechnology that will involve students from at least 4 colleges and 10 university departments. Ten trainee slots are requested for the next training period, which will be augmented by 4 slots funded from university resources. The program requirements include completing: (1) a graduate level, laboratory minor in molecular biotechnology; (2) an off-campus industrial internship; (3) a capstone biotechnology design course; (4) a course in professional development; (5) a course in research ethics; (6) an annual research symposium; and, (7) a biotechnology-related service project. These requirements are in addition to those associated with the student?s particular department or program for the doctoral degree. This program will also provide a central focus for faculty of the various disciplines involved in this training effort to seek out new opportunities for formal and informal research collaboration.
Despite the accelerating expansion of online resources for modern life science education and training, the reality is that development of molecular biology laboratory-based skills requires ???????????????hands-on?????????????????? instruction, preferably in a research-oriented context. Keeping pace with recent scientific breakthroughs and incorporating such developments into the educational setting is challenging from both workforce and economic perspectives. Yet, educational strategies that rely on traditional, lecture alone or outdated, laboratory experiences ineffectively prepare the current and future generations of the US biomedical workforce. Here, we propose to develop and implement a new paradigm for teaching modular molecular biology-oriented laboratory courses that relate established critical skills to cutting-edge technologies in support of the professional viability of modern biologists and biotechnologists. Our overarching goal is to initiate and support a collaborative effort to ???????????????teach the teachers?????????????????? how to Designate, Design, Develop, Deploy and Disseminate (5-D) Molecular Biotechnology Laboratory Education Modules (MBLEMs) on cutting-edge topics across the higher education landscape. This aim will be accomplished by fostering multi-institution cooperation, creative new pedagogical approaches, and integration of interdisciplinary research and bioethics into novel educational modules. We will collaborate initially with teams of participants from five different institutions, and then add three new partners along the way, that collectively represent a broad range of higher educational missions. In support of the MBLEM effort, we will establish a virtual support network, i.e., ongoing inter-institution communication throughout the year to enable MBLEM implementation, led by faculty and teaching postdoctoral fellows at North Carolina State University (NC State). The year-long, 5-D support process will be centered around an annual summer workshop at NC State attended by representatives of our partner institutions that will nucleate interdisciplinary teams of faculty, teaching assistants (graduate students and postdoctoral fellows) and experienced MBLEM instructors. As part of this process, we will organize career development and outreach opportunities that bring together faculty and students from the partner institutions, including those that focus on serving minority students and students with disabilities, to form a supportive and inclusive community fostering modern life science laboratory training. We will also assess MBLEM dissemination and the impact of training on participating MBLEM institutions educational programs and students. Assessment will enable the identification of challenges faced at different institutions and reveal how to better train diverse groups of students in critical skills needed to be productive members of the US biomedical workforce.
Through our GAANN Fellowship Program in Molecular Biotechnology, North Carolina State University (NC State) proposes to increase its already strong commitment to graduate training in aspects of molecular biotechnology through its Molecular Biotechnology Training Program (MBTP), enriching disciplines designated by GAANN as critical to national needs. Our Program will ensure technical proficiency and training in responsible and rigorous science for 8 GAANN Fellows supported by DoEd and 3 additional Fellows supported by NC State.
Most microorganisms utilize organic carbon in its various forms (carbohydrate, protein, fats) as primary sources of food and energy. However, certain microorganisms (extremophiles), perhaps as a nod to their primordial origins, have another way. They utilize inorganic solid materials in the absence of light to gain energy for cell biosynthesis and maintenance, an unusual biological characteristic, referred to as chemolithotrophy. However, this physiological mode is common among microorganisms known as extremely thermoacidophilic archaea (Topt > 70?????????C, pHopt < 3.5). Not only do these microorganisms live in hot acid, they also utilize minerals and elemental sulfur for energy to drive cellular processes. The mechanisms underlying the acquisition of electrons from metals and sulfur have important biological and biotechnological implications. We have developed specialized methods to study the growth physiology of extreme thermoacidophiles in the lab and have expanded the genomics database to now includes over 25 whole genome sequences and related ???????????????omics?????????????????? information. We have also incorporated genetic tools, further expanding the prospects for understanding the basis of chemolithotrophy in hot, acidic biotopes. Extreme thermoacidophiles provide unique insights into how biotic and abiotic phenomena interact to drive metal heavy and sulfur oxidation through their novel biomolecular machines that include membrane oxidase complexes. These complexes coordinate the acquisition of electrons from solid forms of iron (and other metals, such as vanadium, molybdenum and uranium) and sulfur that ultimately lead to the generation of cellular energy and reducing power in the form of ATP and NADH. In some cases, these processes are linked to the fixation of CO2, thereby also powering autotrophy and obviating the need for organic carbon altogether. One important aspect of this project is the establishment of non-model, extremophilic microorganisms as platforms for biology and biotechnology. Most applications to date center on well-studied, heterotrophic microbes (e.g., Escherichia coli, Saccharomyces cerevisiae), even though there are many interesting (and technological promising) biological phenomena that remain unstudied, as is the case with extremophiles. Here, we focus on fundamental aspects of chemolithotrophy, with an eye towards insights that have scientific and biotechnological significance. In the broader sense, by understanding the intrinsic mechanisms driving extremophily, we will expand the range of conditions under which biological systems and molecules can function for biotechnological purposes.
Herein, we plan to use systems biology-guided approaches to develop two non-model, microbial metabolic engineering platforms based on the most thermophilic crystalline cellulose-degrading organism known, the bacterium Caldicellulosiruptor bescii (Cbes), which grows up to 90?????????C, and the most thermophilic sugar-utilizing organism known, the archaeon Pyrococcus furiosus (Pfu), which grows up to 103?????????C. This work leverages recent breakthrough advances in the development of molecular genetic tools for these organisms, complemented by a deep understanding of their metabolisms and physiologies gained over the past decade of study in the PIs?????????????????? labs. We will apply the latest metabolic modeling approaches to optimize biomass-to-product conversion using biomass, such as corn fiber and switchgrass, to produce 3-hydroxypropionate (3HP), 1-propanol, succinate and 1,4-butanediol as example products. Cbes and Pfu have already been metabolically engineered at NCSU and UGA to convert biomass-derived sugars and CO2 into bioproducts, such as ethanol, 3HP, n-butanol and acetone, with enhanced deconstruction capacity, e.g. [1-8]. The over-arching goal is to demonstrate that two non-model microbes, specifically extreme thermophiles, can be strategic metabolic engineering platforms for industrial biotechnology.
Challenges at the FEW nexus are not simply technological, but convergent in the sense of spanning technical, ecological, social, political, and ethical issues. The field of biotechnology is evolving rapidly - and with it, the potential for creating a diverse array of powerful future products that could intentionally and unintentionally impact FEW systems. Depending on what products are developed and how those products are deployed, biotechnology could have a positive or negative impact on all 3 of these systems. Wise decisions will require leaders who can integrate knowledge from engineering, design, natural sciences, and social sciences. We will train STEM graduate students to respond to these challenges by conducting convergent research aimed at development, and assessment of biotechnologies to improve services provided by FEW systems. We will train our students to engage with non-scientists to elevate societal discourse about biotechnology. We will recruit 3 cohorts with emphasis on students who have shown a passion for crossing between natural and social sciences. We will work with the NCSU Initiative for Maximizing Student Diversity in recruiting students from underrepresented minority groups. Cohorts will have 6 students who will take a minor in Genetic Engineering and Society (GES). They will receive PhDs in established graduate programs such as Plant Biol, Chem & Biomol Engr, Econ, Public Adm, Entomol, Plant Path, Communication, Rhetoric & Digital Media, Forestry & Environ Res, Crop & Soil Sci, and Genetics. For students in natural science PhD programs, at least 1 thesis committee member will be from a social sciences program and vice versa for students in social sciences. For all students, at least 1 thesis chapter will demonstrate scholarship across natural and social sciences. The disciplinary breadth of our proposed NRT is very broad, so we will focus student projects narrowly on a specific biotechnology product that impact FEW systems. When they first arrive at NCSU, cohorts will participate in a training program off campus where they will be exposed to the issues they will address. Students will carry out a group project in the focus area of the cohort to continue team development. To fulfill the GES minor, students will take 3 specially designed courses: Plant Genetics & Physiology, Science Communication & Engagement, Policy & Systems Modeling. There are no NRT-eligible institutions partnering on this project outside of an evaluation role.
Biomanufacturing differs from chemical manufacturing as the process operations are significantly different in deference to the lability of biomolecules and cells. Biomanufacturing also differs in the expertise needed for designing, developing and implementing bioprocesses as well as the nature of safety and ethical issues that must be addressed. In the nascent industrial biotechnology sector, the pace of change and innovation, along with societal impacts, must be part and parcel of workforce training and education. Rather than develop separate educational programs for molecular biotechnology, bioprocessing and the ethical issues related to the field, we propose to provide an integrated platform, based on the best pedagogical practices and educational technologies (e.g., including the use of augmented reality for remote laboratory training) that brings workers up-to-speed and helps them maintain the needed expertise to be effective in this emerging sector. BIT (https://biotech.ncsu.edu/), BTEC (www.btec.ncsu.edu) and GES (https://research.ncsu.edu/ges/) at NC State have considerable experience in this type of education for our campus and beyond, and propose to leverage this experience to contribute to the BioMADE initiative. This integrated educational training will help build a sustainable, domestic, end-to-end bioindustrial manufacturing ecosystem that will enable domestic bioindustrial manufacturing at all scales, develop technologies to enhance U.S. bioindustrial competitiveness, de-risk investment in relevant infrastructure, and expand the biomanufacturing workforce to realize the economic promise of industrial biotechnology. Recent attention to issues of Diversity, Equity, and Inclusion (DEI), and broader societal awakenings of academic and corporate responsibility have raised important questions that reach well beyond our laboratories, classrooms, manufacturing facilities, and into society. The current and future biomanufacturing workforce, need to be prepared for these complexities. The workforce training and education package developed here will be sensitive to student/worker time commitment and be maintained such that emerging developments and innovations can be readily incorporated.
Title: Systems Biology-Based Optimization of Extremely Thermophilic Lignocellulose Conversion to Bioproducts Principal Investigator: Michael. W.W. Adams Job Title: Professor Institution: University of Georgia PI Phone Number: 706 542 2060 PI Email Address: adamsm@uga.edu FOA Number: DE-FOA-000186510 Participating Investigators: Ying Zhang, Univ. of Rhode Island Dmitry A. Rodionov, Sanford-Burnham-Prebys Med. Dis. Institute Robert M. Kelly, North Carolina State University Proposed Technology. Herein, we plan to use systems biology-guided approaches to develop a non-model, microbial metabolic engineering platform based on the most thermophilic cellulose-degrading organism known, Caldicellulosiruptor bescii, which grows optimally at 78?????????C. This work leverages recent breakthrough advances in the development of molecular genetic tools for this organism, complemented by a deep understanding of its metabolism and physiology gained over the past decade of study in the PIs' labs. We will apply the latest metabolic modeling approaches to optimize biomass to product conversion using switchgrass as the model plant and acetone and 3-hydroxypropionate as products. The over-arching goal is to demonstrate that a non-model microorganism, specifically an extreme thermophile, can be a strategic metabolic engineering platform for industrial biotechnology. PROJECT GOALS: Aim 1. To construct and test a robust metabolic model of C. bescii. This will include the degradation and assimilation of C6 and C5 polysaccharides, sugar transporters and transcriptional regulators, intracellular redox pools (NAD/H, NADP/H and reduced/oxidized ferredoxin), and heterologously-expressed genes for product formation. Aim 2. To optimize plant biomass degradation guided by metabolic modeling. This will include regulating expression of key native and non-native glycoside hydrolases in coordination with carbohydrate transporters. Aim 3. To optimize product generation guided by metabolic modeling. This will involve balancing the supply of redox cofactors to match the requirements for acetone and 3-hydroxypropionate production and regulation of the expression of key oxidoreductases and also of their activities through post-translational modification. Aim 4. To demonstrate conversion of cellobiose, microcrystalline cellulose and switchgrass to bio-products by engineered C. bescii strains for target products at bioreactor-scale. This will involve testing recombinant C. bescii for deconstruction and conversion to acetone and 3-hydrolypropinate in continuous and semi-batch culture. STATEMENT OF WORK FOR NCSU: NCSU will take the Lead on Aims 2,4 and also participate in accomplishing Aims 1,4. Details on the technical approaches to be used are detailed in the proposal narrative.
Overview: Extremely thermoacidophilic archaea are metabolically versatile microorganisms belonging to the order Sulfolobales that thrive in hot acid (Topt 75?????????C, pHopt < 3.5). Some species can grow chemolithoautotrophically, based on a novel CO2 fixation cycle that includes 3-hydroxypropionate and 4-hydroxybutyrate as intermediates (3HP/4HB cycle). Species within the Sulfolobales oxidize reduced inorganic sulfur compounds (RISCs) for bioenergetic benefit. Recently, molecular genetic tools have become available for several species in the genus Sulfolobus, such that in-frame markerless deletions and chromosomal insertions are possible. As a result, extremely thermoacidophilic archaea could serve as metabolic engineering platforms, thereby expanding current options for industrial biotechnology by making use of inorganic carbon (i.e., CO2) and energy (i.e., sulfur) sources that otherwise are not utilized. Intellectual Merit: The objective for the proposed work is to create metabolically engineered strains of the model thermoacidophilic archaeon, Sulfolobus acidocaldarius DSM639, that produce chemicals from CO2 and RISCs. Pan genomic analysis of the Sulfolobales has revealed the presence of a CO2 fixation cycle and key genes that can be recruited to enable S. acidocaldarius to oxidize RISCs. Bio-based chemical production from CO2 that specifically leverages biological function in hot, acidic conditions will be examined from engineering perspectives, informed by fundamental assessments of wild-type and recombinant S. acidocaldarius physiology. Extremophile metabolic engineering to exploit their unique biological characteristics is an untapped, transformative opportunity for industrial biotechnology. 1. Create and then characterize metabolically engineered S. acidocaldarius DSM639 strains that utilize CO2 as a carbon source and RISCs as energy sources. S. acidocaldarius encodes a cycle for CO2 fixation and an incomplete pathway for sulfur oxidation that can be repaired with genes recruited from chemolithoautotrophic Sulfolobales. 2. Demonstrate that a functional, biosynthetic, thermophilic pathway for acetone can be engineered into strains of S. acidocaldarius DSM639. We have identified genes/enzymes to be recruited from other thermophilic microorganisms that will enable S. acidicaldarius to produce an industrial chemical from simple sugars. 3. Demonstrate that engineered strains of S. acidocaldarius DSM639 can produce acetone from CO2 as a carbon source and RISCs as energy sources. The goal is to obtain acetone production under chemolithoautotrophic conditions. 4. Establish at bioreactor scale that engineered S. acidocaldarius DSM639 strains can produce acetone that can be recovered through in situ distillation. T-x-y information indicates that acetone-water mixtures form a two-phase system at aqueous acetone concentrations that will facilitate recovery as a process intensification step. Broader Impacts: Expanding opportunities for metabolic engineering into non-model host microorganisms will be pivotal is tapping biodiversity to the fullest extent possible. During each year of the award, our lab will also work with faculty and students at the North Carolina School for the Deaf (NCSD) to develop biotechnology-based teaching modules that incorporate close-captioning and other features to facilitate educational objectives for the hearing-impaired. Examples derived from the research proposed here on extremophiles will be featured in these modules. In addition to on-site demonstration of these modules at NCSD each year, the NCSU Biotechnology (BIT) Program (RMK is the Director) will host visiting middle school and high school students from NCSD annually for a 2-day laboratory experience to take advantage of our facilities and educational methods (www.ncsu.edu/biotechnology). Furthermore, we will partner with the National Technical Institute for the Deaf (NTID) and the Southeast Regional STEM Center (SRSC) to bring together 7-12 grade educators for the deaf and hard-of-hearing for leveraging STEM education re
Corn fiber represents an underused component of the plant with respect to its conversion to biofuels and biochemicals. High levels of carbohydrate release from the fiber matrix are thwarted by the recalcitrant nature of lignocellulose. Enzymatic treatments with conventional cellulase and hemicellulase cocktails are not effective in achieving nearly full recovery of fermentable sugars. The genus Caldicellulosiruptor contains extremely thermophilic bacteria (Topt > 70*C), including many species which are highly cellulolytic and hemicellulolytic. The pan genome for this genus encodes a wide range of novel glycoside hydrolases (GHs), some of which have been characterized biochemically and some that are not yet studied, that hold promise for the extensive degradation of corn fiber. In the proposed project, representative cellulases and hemicellulases from Caldicellulosiruptor species will be produced recombinantly in either Escherichia coli or Caldicellulosiruptor bescii for evaluation against corn fiber targets. In addition, transcriptomic screening will be carried out for Caldicellulosiruptor species during growth on the corn fiber to tease out genes encoding enzymes of potential importance and to identify synergistic interactions among secretome-bound GHs. The overarching goal of the project is to identify extremely thermophilic GHs from Caldicellulosiruptor species that are capable of extensive conversion of corn fiber into fermentable sugars.