(2ab) Belowground Carbon Farming: Engineering Genetic Circuits in Plant Roots and Rhizobacteria for Soil Carbon Sequestration

Climate change effects caused by increasing atmospheric carbon dioxide levels have made mitigation efforts critical for humanity in the 21st century. A key strategy proposed to aid carbon sequestration is engineering plants to increase transfer of photosynthesis-fixed carbon into soils. While release of organic exudates (primary metabolites/biopolymers) by plant roots and their utilization by root-associated bacteria (rhizobacteria) is the primary mechanism for transfer of plant-derived carbon into soils, the underlying plant/bacterial genes and metabolic pathways that control this transfer remain unclear. The goal of my research is to use synthetic biology to elucidate these fundamental processes and develop genetic toolkits for engineering belowground carbon flux. This information can be applied to design synthetic root systems and biofertilizer bacteria that augment soil carbon storage, increase bioenergy/food crop productivity, and convert root-derived carbon into agronomically-relevant chemicals.

Research Interests

Graduate Work

My graduate research centered on engineering specialized redox pathways of soil bacteria that enable genetic control over materials science applications. Specifically, I built genetic circuits to tune respiratory electron flux within the model electroactive bacterium, Shewanella oneidensis, which is able to transfer electrons onto extracellular electron acceptors (iron, palladium, electrodes) during anaerobic growth. This capability allowed me to create genotype-phenotype linkages between expression of key electron transfer proteins and abiotic technologies, such as pollutant remediation, power generation, and metal-catalyzed synthesis reactions. By building genetic circuits that could toggle electron flux on/off based on the presence of chemical stimuli and key gene expression regulators, I was able to optimize the magnitude and spatiotemporal dynamics of electron flux by engineered strains. These circuits were used to genetically control the synthesis of macroscopic polymer and hydrogel materials, which are traditionally limited to synthesis via non-biological means. My work demonstrated that extracellular electron transfer is a powerful modality to connect bacterial physiology with abiotic chemistry and that this respiratory mechanism is controllable using synthetic biology.

Postdoctoral Work

My current research bridges plant and bacterial synthetic biology to engineer transfer of the primary photosynthetic sugar product, sucrose, between roots systems and soil microbiomes. While sucrose delivery to non-photosynthetic root tissues is a strong controller of root biomass, soil microbial activities, and belowground carbon input, there are few genetic tools developed in plants/rhizobacteria to monitor or optimize this key interkingdom carbon exchange. Using the rhizobacterial and bioindustrial chassis, Pseudomonas putida, I built fluorescence-outputting genetic sensor circuits that spatiotemporally measure root-released sucrose and applied metabolic engineering to enable bacterial growth with sucrose as a carbon source. By pairing these constructs with the model plant Arabidopsis thaliana, I probed how sucrose exudation is affected by plant genetics and environmental stresses, modulates plant-bacteria interactions, and can serve as carbon input for root-colonizing biofactories within soil microbiomes. Furthermore, engineered rhizobacteria enabled rapid phenotyping of synthetic plant metabolism circuitry that elevates sucrose exudation and delivery to target bioinoculants. To uncover gene-and tissue-level design rules for exudate reprogramming, I built genetic circuits within Nicotiana benthamiana and A. thaliana that tune spatial and magnitudinal patterns of plant cell sucrose release via differential enzyme and transporter expression. Sucrose levels of transgenic plants were subsequently interrogated by bacterial biosensor-driven fluorescence to pinpoint plant genetic architectures that maximize root exudation. My results have shown how bacterial genetic circuitry can be used to engineer agriculturally-relevant microbiomes and aid plant synthetic biology design-build-test-learn cycles for soil carbon-sequestering traits.

Future Work

As a faculty member, my research group will build upon my graduate (soil bacteria synthetic biology) and postdoctoral training (plant synthetic biology) to address two key stages in the mass balance of carbon flow through root-bacteria-soil systems: (Aim 1) boosting the flux of carbon from roots to soil bacteria and (Aim 2) enabling the conversion of plant-fixed carbon into stable storage compounds and functional agrochemicals within soil. While my postdoctoral work has demonstrated that manipulating plant/bacterial sucrose metabolism is an advantageous handle for augmenting root-release of carbon, sucrose itself is a relatively promiscuous environmental substrate that is consumed by many microbes. To ensure high yield conversion of plant-derived carbon into stable or functional target compounds, my laboratory will create networks of ‘biologically insulated’ sucrose flux between plant roots and soil bacteria partners (Aim 1). This will be accomplished by expressing previously characterized enzymes and transporters in plants/bacteria to convert sucrose into derivative sugars (isomers, esters) that are less metabolically available to competing microbiota. We will determine whether these sucrose analogs can be produced by transgenic plants at high titers and if they can serve as biomass building block molecules for cognate rhizobacteria. My group will also engineer the biological production of polymers that stabilize root-exuded carbon within soil systems and confer beneficial traits to crops, such as drought/salinity tolerance (Aim 2). Within transgenic roots and rhizobacteria, we will express biosynthetic pathways for key polymers (polyhydroxyalkanoates, suberin, long chain fatty acids) under the control of root metabolite/hormone sensors to spatiotemporally program deposition of these biomolecular barriers in patterns that maximize plant yields and agronomic phenotypes. These technologies will be initially prototyped in rhizobacteria (P. putida, Bacillus subtilis) and plant models (N. benthamiana, A. thaliana, Setaria viridis) and later moved into Department of Energy- and US Department of Agriculture-relevant species, such as environmental bacterial isolates, maize, and sorghum. More broadly, my group will establish how chemical engineering and synthetic biology design principles can be applied toward climate change-relevant challenges in plant and agricultural science.

Teaching Interests

As a professor, I will be an enthusiastic teacher to students and trainees in both the laboratory and classroom. My undergraduate/graduate education in chemical engineering has prepared me to teach thermodynamics, chemical kinetics, and transport phenomena courses, where I could infuse my interest in biology with these topics. For example, as a teaching assistant for undergraduate and graduate kinetics classes, I aided students’ conceptual and practical understanding of material by creating problem sets with enzymatic and cellular systems. As a postdoc, I similarly developed a lecture for a biology special topics course where undergraduates learned genetic circuit design through a hands-on activity with “LEGO biobricks” of genetic parts. I have also enhanced my pedagogy skills by attending workshops at Stanford that aid postdocs in inclusive course design and teaching behaviors. As a faculty member, I will build upon these experiences to emphasize plant science as an emerging area within chemical engineering education. For example, metabolite mass balances, enzyme kinetics, and fluid flow are central to studying/engineering plants and are topics that chemical engineers are adept to tackle. I will incorporate plant-based examples of these areas within the core chemical engineering curriculum and develop special topics classes that cover more advanced phenomena on plant genetic engineering. Agriculture is a unifying vocation across many socioeconomic, racial, and ethnic groups that are underrepresented within STEM, and expanding chemical engineering training to encompass aspects of plants might aid inclusion of these minorities. Formalized communication of plant science findings with farmers and the broader public is commonplace within agriculture research (termed extension), and I would similarly perform this outreach along with graduate students and trainees in my research group. Collectively, my role as an educator would be to advance student understanding of chemical engineering fundamentals and aid their identification of new fields/problems (agriculture, sustainability) where this knowledge can be applied.

Postdoctoral Project: Developing Design Rules for Sucrose Exchange Between Roots and Soil Bacteria

Postdoctoral Mentor: José R. Dinneny, Department of Biology, Stanford University

PhD Dissertation: Harnessing Bacterial Electroactivity with Materials Science and Synthetic Biology

PhD Mentor: Benjamin K. Keitz, McKetta Department of Chemical Engineering, University of Texas at Austin

Fellowships/Awards

Stanford TomKat Center Postdoctoral Fellowship in Sustainable Energy, 2020

UT Austin Graduate School Continuing Fellowship, 2019

NSF Graduate Research Fellowship (Honorable Mention), 2015

Barry M. Goldwater Scholarship, 2013

Highlighted Publications (*Equal Contribution)

Dundas, C.M., Dinneny, J.R (2022) Genetic Circuit Design in Rhizobacteria. BioDesign Res. 2022:9858049.

Graham, A.J.*, Partipilo, G.*, Dundas, C.M., Miniel Mahfoud, I.E., FitzSimons, T., Rinehart, R., Chiu, D., Tyndall, A.E., Rosales, A.M., Keitz, B.K. (2021) Transcriptional Regulation of Synthetic Polymer Networks. bioRxiv. DOI: 10.1101/2021.10.17.464678.

Alcalde, R.E., Dundas, C.M., Dong, Y., Sanford, R.A., Keitz, B.K., Fouke, B.W., Werth, C.J. (2021) The Role of Chemotaxis and Efflux Pumps on Nitrate Reduction in the Toxic Regions of a Ciprofloxacin Concentration Gradient. ISME J. 15:2920-2932.

Dundas, C.M., Walker, D.J.F., Keitz, B.K. (2020) Tuning Extracellular Electron Transfer by Shewanella oneidensis Using Transcriptional Logic Gates. ACS Synth. Biol. 9(9):2301-2315.

Graham, A.J., Dundas, C.M., Hillsley, A., Kasprak, D.S., Rosales, A.M., Keitz, B.K. (2020) Genetic Control of Radical Cross-linking in a Semi-Synthetic Hydrogel. ACS Biomater. Sci. Eng. 6(3):1375-1386.

Springthorpe, S.K., Dundas, C.M., Keitz, B.K. (2019) Microbial reduction of metal-organic frameworks enables synergistic chromium removal. Nat. Commun. 10:5212.

Dundas, C.M., Graham, A.J., Romanovicz, D.K., Keitz, B.K. (2018) Extracellular Electron Transfer by Shewanella oneidensis Controls Palladium Nanoparticle Phenotype. ACS Synth. Biol. 7(12):2726-2736.

Fan, G.*, Dundas, C.M.*, Graham, A.J., Lynd, N.A., Keitz, B.K. (2018) Shewanella oneidensis as a living electrode for controlled radical polymerization. Proc. Natl. Acad. Sci. U.S.A. 115(18):4559-4564.