(6ir) Engineering a Purple Non-Sulfur Bacterium to Expand Symbiotic Nitrogen Fixation

When bio-available nitrogen is limited, plant growth and thus crop yields are reduced. Synthetic fertilizers have answered this need, nourishing crops that feed nearly 50% of the world’s current population. While synthetic fertilizers have largely enabled the Green Revolution, their production and use have introduced new problems. Five percent of the natural gas and two percent of the energy produced in the world annually are consumed to make ammonia. When the fertilizer is applied to the soil, microbes convert it to nitrate. Plants can utilize nitrate, but it is easily washed into the water supply where it can exceed safe drinking water limits. Nitrate accumulation in waterways also contributes to hypoxic marine dead zones. Many prokaryotes use the enzyme nitrogenase to fix their own nitrogen and some form symbiotic associations with plants, greatly reducing the plant’s need for soil nitrogen. Expanding symbiotic nitrogen fixation to a larger number of crop species is the motivation for my research.

Developing nitrogen-fixing chloroplasts could support a plant’s nitrogen requirements. Expressing the nif cluster, the genes required to produce nitrogenase, from a diazotrophic cyanobacterium in a non-diazotrophic cyanobacterium could help uncover the requirements of nitrogen fixation in a photosynthetic host, including chloroplasts which evolved from a cyanobacterium. My graduate work focused on creating synthetic regulators for the predictable and specific expression of the nif cluster in the model non-diazotrophic Synechocystis sp. PCC 6803. When expressing genes in a new host, the native regulation that protected the proteins or minimized the required cellular resources is often missing. I developed genetic circuits to control transcription of the nif cluster in response to environmental conditions important for the function of nitrogenase, including oxygen, nitrogen, and light. To begin to mimic the complexity of gene expression in the native host, I created regulators that incorporated a feed forward loop and others that responded to two inputs with AND logic, in addition to single-input (inducible) promoters. I replaced the regulatory region between divergent genes in the nif cluster with oxygen-responsive promoters that increased nitrogenase activity by 50% in Synechocystis. This construct could also be used to probe the cluster for unidentified promoters.

My post-doctoral research has focused on engineering the metabolically versatile purple non-sulfur bacterium (PNSB) Rhodopseudomonas palustris CGA009 to utilize its ability to convert waste products, such as depolymerized lignin and carbon dioxide, into value-added compounds, including polyhydroxybutyrate and putrescine. I am currently developing synthetic regulators of gene expression since only one orthogonal, constitutive promoter has been used for gene expression in published literature, and it has not even been characterized. My work includes expanding an inducible promoter’s dynamic range, testing a second orthogonal inducible system, generating an RBS library to expand and tune expression, and increasing the bacterium’s sensitivity to selection pressure. I am also characterizing the range of lignin-derived compounds photosynthetic, nitrogen-fixing R. palustris can degrade and the metabolic pathways it uses for that degradation.

My future research interests focus on engineering R. palustris to form nitrogen-fixing nodules in a legume that already establishes a symbiotic relationship with one of the PNSB’s close phylogenetic relatives. R. palustris can be found in the tropical rainforest rhizosphere and is known to promote plant growth. This PNSB is phylogenetically close to both Bradyrhizobium japonicum, which forms nitrogen-fixing root nodules in soybeans, and the photosynthetic stem-nodulating Bradyrhizobium sp. ORS278; yet, R. palustris has not been found in endosymbiotic relationship with any plant. Most research on the symbiotic relationship between legumes and nitrogen-fixing bacteria have focused on Nod factor-dependent plants or an existing symbiotic bacterium. Engineering a bacterium with many of the genes, proteins, and traits that are common among the stem-nodulating Bradyrhizobia but that is not an endosymbiont could uncover the crucial steps needed to achieve nitrogen-fixing endosymbiosis, first in the Nod factor-independent Aeschynomene evenia. Using the synthetic biology tools that I am currently developing, I will begin by expressing genes from two horizontally-acquired islands present in the photosynthetic stem-nodulating Bradyrhizobia, but not R. palustris. Another early project will be the introduction of mevalonate production, which is known to induce calcium spiking and the expression of symbiotic genes in vascular plants that form an endosymbiotic relationship with mycorrhizal fungi or rhizobial bacteria. This work could make significant contributions to the expansion of nitrogen-fixing symbiosis in crops.

Successful Proposals:

University of Nebraska Office of Research and Economic Development Revision Award, University of Nebraska Core Facility Grant Program for New Users Award, Nebraska Corn Board, NSF Graduate Research Fellowship, People, Prosperity and the Planet Student Design Competition for Sustainability

Postdoctoral Project:

"Harnessing the metabolically versatile Rhodopseudomonas palustris as a model bacterium"

Under the supervision of Dr. Rajib Saha, Department of Chemical and Biomolecular Engineering, University of Nebraska

PhD Dissertation:

“Genetic Circuits for Transcriptional Regulation in Synechocystis sp. PCC 6803”

Under the supervision of Dr. Tae Seok Moon, Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis

Selected Publications:

CM Immethun and TS Moon. in Synthetic Biology of Cyanobacteria Ch. 13. Synthetic Gene Regulation in Cyanobacteria (Springer/Nature Press, 2018).

CM Immethun, DM DeLorenzo, CM Focht, D Gupta, CB Johnson, TS Moon. Physical, Chemical and Metabolic State Transcriptional Regulators Expand the Synthetic Biology Toolbox for Synechocystis sp. PCC 6803, Biotechnol Bioeng. 114:7 (2017).

N Wan, DM DeLorenzo, L He, L You, CM Immethun, G Wang, EEK Baidoo, W Hollinshead, JD Keasling, TS Moon, YJ Tang. Cyanobacterial Carbon Metabolism: Fluxome Plasticity and Oxygen Dependence, Biotechnol Bioeng. 114:7 (2017).

CM Immethun, KM Ng, DM DeLorenzo, YC Lee, B Waldron-Feinstein, TS Moon. Oxygen-Responsive Genetic Circuits Constructed in Synechocystis sp. PCC 6803, Biotechnol Bioeng. 113:2 (2016).

Teaching Interests:

Knowledge acquired in college covers only a small part of what an engineer will need to know throughout their career. It provides the foundation from which we start solving a problem; yet, the broad, complex and dynamic situations engineers face requires the confidence to think creatively and the ability to direct our own learning. Students’ development of a firm knowledge base from which they have the confidence to solve the variety of problems they will face throughout their careers is my first educational objective. My students will tackle realistic problems designed to increase their comfort with resolving underdetermined systems, addressing the complex web of needs and constraints, thinking creatively, and communicating effectively. Practicing an organized and dynamically controlled thought process in new learning situations is the second educational objective for my students. By assessing the task at hand, evaluating the available resources, planning the approach, applying and monitoring the planned strategies, reflecting on the success of the planned strategies, and revising by adjusting the approach, securing additional resources and sometimes even re-assessing the task, students become self-directed learners. My courses utilize goal-directed practice with targeted feedback to help them embrace the learning process and realize its utility in solving the problems they will encounter. I returned to academia propelled by the desire to help students construct the foundation from which they can make important contributions to the challenges we face as a society. My instructional strategies and assessment methods are designed to support that goal and my educational objectives.

Selected Teaching Experience:

Teaching Assistant, Chemical Process Dynamics and Control - Received the EECE Graduate Student Teaching Award, based on student evaluations

Advisor, International Genetically Engineered Machine (iGEM) Competition - Washington University in St. Louis and University of Nebraska teams

Mentor, 11 Graduate Rotation and Undergraduate Students, Washington University in St. Louis - 7 are co-authors on my publications

Judge, International Genetically Engineered Machine (iGEM) Competition's Giant Jamboree

Teacher Workshop Instructor, BioBuilder^® Educational Foundation