Engineering a Carbon Dioxide Concentrating Mechanism in Cupriavidus necator for Carbon-Negative Biomanufacturing
Authors:
Akira Nakamura1,2* ([email protected]), Farren J. Isaacs1,2,3, Michael Jewett4
Institutions:
1Department of Molecular, Cellular, and Developmental Biology, Yale University–New Haven; 2Systems Biology Institute, Yale University–New Haven; 3Department of Biomedical Engineering, Yale University–New Haven; 4Stanford University–Palo Alto
Goals
The program goal is to develop high-throughput biosystems design tools in carbon dioxide (CO2)-fixing biosystems and apply these tools to engineer biosynthetic pathways for carbon-negative biomanufacturing of simple commodity chemicals. In this project, researchers aim to increase the CO2 utilization efficiency of the CO2-fixing microbe, Cupriavidus necator, by reconstituting the α-carboxysomal CO2 concentrating mechanism native to Halothiobacillus neapolitanus.
Abstract
The accelerating climate crisis combined with rapid population growth poses some of the most urgent challenges to humankind. A major contributing factor to this crisis is the unabated release and accumulation of CO2 across the biosphere. Researchers can take advantage of this abundance of available CO2 to transform the way the world produces and uses carbon (C) by engineering CO2– fixing biosystems to produce commodity fuels and chemicals. A CO2-fixing organism that is actively being studied as a C-negative biomanufacturing chassis is the bacterium, Cupriavidus necator. While proficient in producing high titers of metabolic products, wild-type C. necator does not grow optimally at ambient levels of CO2 in comparison to at high CO2 conditions on autotrophic metabolism. Researchers propose to optimize C. necator growth under atmospheric conditions by heterologously expressing the α-carboxysomal CO2 concentrating mechanism (CCM) characterized in Halothiobacillus neapolitanus (Desmarais et al. 2019; Flamholz et al. 2020). To enable stable chromosomal expression of this 20kb H. neapolitanus CCM operon in C. necator, the team developed an inducible landing pad for integrase-mediated integration of synthetic cargo. In this project, the team demonstrates integration of the bacterial luminescence pathway and tunable expression of the pathway from the landing pad in C. necator. Another objective is to encapsulate the native C. necator Rubisco into the carboxysomal structures instead of heterologously expressing the H. neapolitanus Rubsico. C. necator Rubisco has been reported to retain optimal carboxylation rate in aerobic conditions with abundant competing O2, which is an advantageous trait to maintain for aerobic cultivation (Satagopan and Tabita 2016). Researchers have utilized RFdiffusion, a protein design software, to de novo design C. necator Rubisco binding motifs to replace with the H. neapolitanus Rubsico for carboxysomal encapsulation (Watson et al. 2023). The next steps will entail synthesizing and testing these motifs in vitro for binding.
References
Desmarais, J. J., et al. 2019. “DABs are Inorganic Carbon Pumps Found Throughout Prokaryotic Phyla,” Nature Microbiology 4(12), 2204–15. DOI:10.1038/s41564-019-0520-8.
Flamholz, A. I., et al. 2020. “Functional Reconstitution of a Bacterial CO2 Concentrating Mechanism in E. Coli,” ELife 9, 1–57. DOI:10.7554/ELIFE.59882.
Satagopan, S., and F. R. Tabita, F. R. 2016. “RubisCO Selection Using the Vigorously Aerobic and Metabolically Versatile Bacterium Ralstonia Eutropha,” The FEBS Journal 283(15), 2869–80. DOI:10.1111/FEBS.13774.
Watson, J. L., et al. 2023. “De Novo Design of Protein Structure and Function with RFdiffusion,” Nature 620. DOI:10.1038/s41586-023-06415-8.
Funding Information
This research was supported by the DOE Office of Science, BER program, grant no. DE-FOA-0002600.