Ethylene Synthesis By Genetically Modified Rhodococcus Opacus PD630 on Synthetic Human Urine

A. Williams and K. Wawrousek

Deep space exploration necessitates the ability to generate fuels, chemicals, food, and pharmaceuticals off-earth with limited resources, as it would not be feasible to send all potentially necessary materials via spacecraft. To advance toward the goal of engineering microbes to produce materials that may be needed for human life on other planets such as Mars, we are genetically modifying the non-model bacterium Rhodococcus opacus PD630 to produce ethylene, the monomer necessary for polyethylene plastic production. R. opacus PD630 was chosen because this genetically tractable, oleaginous microbe is known for its metabolic plasticity, so it may be able to grow on several organic waste streams. To utilize a nutrient source available off-Earth, we first tested this bacterium for growth on synthetic urine. Monitoring the growth behavior of the bacterium, growth curve studies on Luria-Bertani Broth and synthetic human urine were conducted on R. opacus PD630, quantifying overall culture growth by the change in optical density at 600 nm. Within the synthetic urine growth media, our bacterium entered the stationary phase after 50 hours of incubation at 30°C and shaking at 150 RPM, reaching a final optical density of 1.3. To engineer R. opacus PD630 to produce ethylene, the ethylene forming gene (efe) was codon optimized for Rhodococcus and introduced into the pBAV1K plasmid for expression under the constitutive PsbA promoter. Gene expression will be confirmed via immunoblot, and ethylene production will be measured with gas chromatography, as ethylene is a gas at the 30°C growth temperature.

Additional Key Words: Rhodococcus opacus, genetic modification, ethylene production, human urine, Mars colonization, efe gene.

¹ To be presented at the University of Wyoming’s Undergraduate Research Day in December 2021 via poster and oral presentation.

² Karen Wawrousek, Ph.D. is an Assistant Professor in the Department of Chemical Engineering, University of Wyoming, Laramie WY 82071. Alexandria Williams is an Undergraduate Senior Chemical Engineering Student, Dept. of Chemical Engineering, University of Wyoming, pursuing an MS in Biopharmaceutical Process Engineering.