Metabolic Modeling and Genetic Engineering of Enhanced Anaerobic Microbial Ethylene Synthesis
Authors:
Sarah J. Young1* ([email protected]), Kamal M. Deep1, Mikayla Borton3, Elizabeth Morgan1, Ethan King2, Kelly C. Wrighton3, William R. Cannon2, Justin A. North1
Institutions:
1Department of Microbiology, The Ohio State University; 2Pacific Northwest National Laboratory; 3Department of Soil and Crop Sciences, Colorado State University
Goals
To develop robust and optimized anaerobic ethylene pathways in photosynthetic and lignocellulosic bacteria for high-yield conversion of renewable carbon dioxide (CO2) and lignocellulose into bioethylene. This will be accomplished by:
(1) Bioinformatically mining and experimentally screening methylthioalkane reductase homologs, S-adenosyl-L-methionine hydrolase homologs, and alcohol dehydrogenase homologs from cultivated and uncultivated organisms to identify functional enzymes that enhance ethylene yields.
(2) Constructing and employing predictive systems-level models of ethylene production. This project will use a physics-based Rhodospirillum rubrum model to predict enzymes that participate in competing or supporting pathways and are thus targets for selection studies to increase ethylene yields.
(3) Metabolically engineering bacteria for enhanced, sustained ethylene production from CO2 and lignocellulose. The project will assemble the best-performing genes under control of optimized active transcription elements on a modular DNA fragment in a combinatorial manner with guidance from predictive models (see goal 2).
Abstract
Previously, the research team detailed a pathway in the phototrophic bacterium R. rubrum that produces ethylene in the absence of oxygen from methionine and ATP (North et al. 2020). Traditional ethylene production involves energy-intensive cracking of petroleum fossil fuels to meet the 300 million metric ton annual demand. Thus, a sustainable microbial platform for the renewable production of ethylene is urgently needed. The goal of this project is to optimize this anaerobic ethylene production pathway.
Enzyme Screens:
Methylthio-alkane reductase genes and other nitrogenase-like genes identified from microbial genomes were synthesized and assembled by the DOE Joint Genome Institute DNA synthesis program such that each set contained a NifB, NifH, NifD, and NifK homolog. Sequences were screened for activity in an R. rubrum deletion strain devoid of native methylthio-alkane reductase (North et al. 2020). Sequences from the methylthio-alkane reductase phylogenetic clade were active for reduction of volatile organic sulfur compounds of the form R-S-CH3 if R. rubrum could couple electron transfer from cell redox carriers to the NifH homolog. Strikingly, proteins encoded by nitrogenase-like sequences from other phylogenetic clades of unknown function showed no methylthio-alkane reductase activity and showed no activity toward other methylated compounds like methylated amines, sulfates, or phosphates. Thus, the functions of many nitrogenase-like clades remain unknown.
Physics-based Modeling:
During photoheterotrophic growth on organic substrates, purple non-sulfur bacteria like R. rubrum acquire electrons by multiple means and store them as reduced electron carriers. The ratio of oxidized to reduced electron carriers [e.g., ratio of NAD(P)+:NAD(P)H)] is difficult to predict but essential for thermokinetic modeling and predicting the targeted metabolic engineering needed to increase ethylene synthesis. Using physics-based models that capture mass action kinetics consistent with the thermodynamics of reactions and pathways, a range of redox conditions for photoheterotrophic growth was evaluated (King et al. 2023; Cannon et al. 2024). Modeling results and experimental measurements of macromolecule levels (DNA, RNA, proteins, and fatty acids; North et al. 2020) indicate that cellular redox poise results in large-scale changes in biosynthetic pathway activity. The model, which agrees with experimental measurements of macromolecule ratios of cells growing on different carbon substrates, indicates that the dynamics of nucleotide versus lipid and protein production is likely a significant mechanism balancing cellular oxidation and reduction.
Metabolic Engineering:
In R. rubrum, ethylene production involves 12 genes and 9 primary reactions, plus pathways supporting ATP, Acetyl-CoA, and NAD(P)H synthesis. Previously, the research team identified high performing S-adenosyl-L-methionine (SAM) hydrolases, isomerases, aldolases, and methylthio-alkane reductases that individually increase ethylene production. Combining these elements on a mobile plasmid under modest to high levels of constitutive expression results in a 30,000-fold increase in ethylene yields from 0.01 to 300 µmol/g dry cell weight. When antibiotics are included to retain plasmid selection, yields further increase to 1,000 µmol/g dry cell weight. Additional genetic tools are needed to integrate multiple pathway elements onto the chromosome, as 4 to 5 pathway elements on a plasmid represents the largest plasmid size R. rubrum can stably incorporate.
References
Cannon, W.R., et. al. 2024. “Redox Poise during Rhodospirillum rubrum Phototrophic Growth Drives Large-scale Changes in Macromolecular Synthesis Pathways,” arXiv:2401.04862 [q-bio.MN]. DOI:10.48550/arXiv.2401.04862.
King, E., et al. 2023. “An Approach to Learn Regulation to Maximize Growth and Entropy Production Rates in Metabolism,” Frontiers in Systems Biology 3:981866. DOI:10.3389/fsysb.2023.981866.
North, J. A., et al. 2020. “A Nitrogenase-like Enzyme System Catalyzes Methionine, Ethylene, and Methane Biogenesis,” Science 369(6507), 1094–8. DOI:10.1126/science.abb6310.
Funding Information
This project is supported by DOE’s BER Program under contracts DE-SC0022091 (The Ohio State University) and 78266 (Pacific Northwest National Laboratory).